1 Cellular and Molecular Toxicology Division, National Institute of Health
Sciences, 1-18-1 Kamiyoga, Setagayaku, Tokyo 158-8501, Japan
2 Institut für Molekularbiologie, MHH, 30625 Hannover, Germany
3 Division of Mammalian Development, National Institute of Genetics, SOKENDAI,
Yata 1111, Mishima 411-8540, Japan
* Author for correspondence (e-mail: ysaga{at}lab.nig.ac.jp)
Accepted 27 May 2003
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SUMMARY |
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Key words: Mesp2, Notch signaling, Rostrocaudal patterning, Presenilin, Somite segmentation, Mouse
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INTRODUCTION |
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Studies in zebrafish, chick and mouse embryos have established that the
Notch signaling pathway is essential for somite formation and patterning,
particularly for the establishment of the rostrocaudal segment polarity
(Conlon et al., 1995;
Oka et al., 1995
;
Dornseifer et al., 1997
;
Hrabe de Angelis et al., 1997
;
Wong et al., 1997
;
Kusumi et al., 1998
;
Evrard et al., 1998
;
Zhang and Gridley, 1998
;
del Barco Barrantes et al.,
1999
; Takke and Campos-Ortega,
1999
; Holley et al.,
2000
; Takahashi et al.,
2000
; Koizumi et al.,
2001
; Bessho et al.,
2001
) (reviewed by Saga and
Takeda, 2001
). In fact, many zebrafish and mouse mutants for genes
encoding Notch pathway components exhibit defects in the rostrocaudal polarity
of somites. The Notch signaling is closely linked to the putative molecular
clock mechanism that operates in the PSM, as oscillating genes encode Notch
pathway components and mutations in Notch pathway components also affect
cyclic genes (Palmeirim et al.,
1997
; McGrew et al.,
1998
; Forsberg et al.,
1998
; Jiang et al.,
2000
; Holley et al.,
2002
; Oates and Ho,
2002
). The generation of the rostrocaudal polarity is also thought
to be controlled by the molecular clock. However, the precise nature of the
molecular clock is not yet known at all. In zebrafish, defects in the
rostrocaudal polarity are often not distinguished from defects in the
molecular clock function, because most of Notch pathway mutants in zebrafish
exhibit similar phenotypes. For example, zebrafish aei, des and
bea mutant embryos commonly show a salt-and-pepper (randomized)
expression pattern of the rostral- or caudal-half marker genes, instead of
normal regular stripes (Jiang et al.,
2000
; Holley et al.,
2002
). This phenotype is virtually indistinguishable from the
phenotype seen in the her1- and her7-Morpholino-injected
embryo, which shows disruption of cyclic gene expression
(Oates and Ho, 2002
). Thus,
there is no available Notch pathway mutant in zebrafish that enables further
analysis of the mechanism of rostrocaudal patterning separately from the
molecular clock.
By contrast, Notch pathway mutants in mouse exhibit various patterns of
phenotypes regarding the rostrocaudal polarity of somites. For example, in
Delta-like 1 (Dll1)- and RBPjk-null embryos, somites show neither rostral nor
caudal property (del Barco Barrantes et
al., 1999), whereas Delta-like 3 (Dll3), lunatic fringe and
Hes7-null embryos show a salt-and-pepper expression pattern of caudal marker
genes (Kusumi et al., 1998
;
Evrard et al., 1998
;
Zhang and Gridley, 1998
;
Bessho et al., 2001
). In our
previous work, we have demonstrated that Mesp2-null and presenilin 1
(Psen1)-null embryos show opposite phenotypes with respect to the rostrocaudal
polarity of somites (Takahashi et al.,
2000
). The Mesp2-null embryo exhibits caudalized somites, i.e.,
the somite loses the rostral-half property, and the whole somite acquires the
caudal-half characteristics. The reverse is true for the Psen1-null embryo.
These observations led us to some fundamental questions: what is the default
state, and how do these genes cooperate to establish rostrocaudal segment
polarity? In some mouse mutants, such as Dll3-null, oscillation of cyclic
genes is disrupted (Dunwoodie et al.,
2002
). However, in Mesp2-null embryos, the rostrocaudal polarity
is disrupted without affecting oscillation of cyclic genes in the posterior
PSM (Nomura-Kitabayashi et al.,
2002
) (Y.T., unpublished). In Psen1-null embryos, oscillation of
cyclic genes in the posterior PSM normally occurs, although the level of
expression is reduced (Koizumi et al.,
2001
). Therefore Mesp2 and Psen1 serve as good tools for exploring
mechanisms of the rostrocaudal patterning independent of molecular clock
function.
Mesp2 is a member of the Mesp family, a group of bHLH transcription
factors, which is expressed in the anterior PSM just prior to somite formation
and is essential for somite boundary formation as well as formation of the
rostrocaudal polarity (Saga et al.,
1997; Nomura-Kitabayashi et al., 2001). We have previously
observed that the rostrocaudal polarity of somites correlates well with the
spatial pattern and the level of expression of the Notch ligand Dll1.
Genetic analyses of Mesp2-null, and Psen1-null mice, and mice carrying an
activated Notch1 in the Mesp2 locus have led us to propose a
model for rostrocaudal patterning, in which two Notch pathways can be active
in the anterior PSM. One is the Psen1-dependent Notch pathway for inducing
expression of Dll1, and the other is the Psen1-independent Notch
pathway for suppressing expression of Dll1. Mesp2 normally suppresses
the Dll1-inducing pathway and potentiates the
Dll1-suppressing pathway in a region corresponding to one presumptive
somite. When Mesp2 expression becomes restricted to the presumptive
rostral half, expression of Dll1 is induced in the presumptive caudal
half by the Psen1-dependent Notch pathway
(Takahashi et al., 2000
).
However, the ligands for these two Notch pathways have not yet been
identified.
In both zebrafish and mouse embryos, at least two Notch ligands (DeltaC and
DeltaD, and Dll1 and Dll3, respectively) are co-expressed in the PSM, and
their expression domains are finally segregated into the rostral or caudal
half of formed somites (Bettenhausen et
al., 1995; Dunwoodie et al.,
1997
; Haddon et al.,
1998
). These expression patterns imply that these ligands do not
have merely redundant functions, but also have distinct roles in somite
patterning and boundary formation. Despite a large number of studies, possible
functional differences between Dll1 and Dll3 signals are not clear. Likewise,
the roles of Psen1, a Notch signal mediator involved in nuclear translocation
of the Notch intracellular domain (De
Strooper et al., 1999
; Struhl
and Greenwald, 1999
; Ye et
al., 1999
), during somitogenesis are not fully understood. If
Psen1 were equally involved in all aspects of Notch signaling, it is puzzling
that the rostrocaudal patterning defects of somites in the Psen1-null embryo
are unique and different from that in any other Notch pathway mutants
(Takahashi et al., 2000
;
Koizumi et al., 2001
). Thus,
to elucidate the precise requirements for Psen1 functions in somite
patterning, further studies are required.
We have conducted genetic studies of the roles in rostrocaudal patterning of Dll1- and Dll3-mediated Notch signaling, the relationships between Notch signaling and Mesp2 function, and the involvement of Psen1 in Dll1- and Dll3-mediated Notch pathways. Our analysis of these genetic interactions revealed several novel findings.
Based on these findings, we propose a new model for stripe formation in the anterior PSM, which is different from the previous hypothesis that rostrocaudal patterning, i.e. formation of the half-a-somite stripe pattern of gene expression, can be regarded as a result of stabilization of oscillating expression in the posterior PSM.
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MATERIALS AND METHODS |
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Analysis of phenotypes
The methods for gene expression analysis by whole-mount in situ
hybridization, histology and skeletal preparation by Alcian Blue/Alizarin Red
staining are as described in previous paper
(Saga et al., 1997). A strong
emphasis was placed on obtaining a precise and accurate comparison of gene
expression patterns and intensity of signals between different genotypes.
Littermate embryos from crosses of double-heterozygous parents were
simultaneously fixed and processed for in situ hybridization. Coloring
reactions in BM purple solution (Roche) were stopped at exactly the same time
for each embryo. To evaluate gene expression precisely in the double mutant
embryo, simultaneous staining of wild-type and single mutant littermates as
controls is essential. Therefore, in all of the images presented in the
figures, the arranged embryos are littermates. At least four, but more usually
six, double-null embryos were used for gene expression analysis with more than
ten single mutants and many more wild-type embryos. Observed differences in
gene expression levels were typically reproduced in triplicate. In the case of
skeletal morphologies, each of eight Dll3/Mesp2 double-null fetuses exhibited
almost complete fusion of neural arches. For vertebral morphologies in
Dll3/Psen1 intercrosses, the number of fetuses is presented in supplementary
data S2F. Each of six Dll3/Psen1 double-null fetuses showed reduced amounts of
disorganized skeletal elements. Whole-mount specimens and skeletal
preparations were observed and photographed with a Leica dissection microscope
equipped with a Fujifilm digital camera (HC-2500) under specific illumination
conditions.
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RESULTS |
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|
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Dll3 and Mesp2 are required for normal expression of each other
Dll3 is the other Notch ligand expressed in the PSM, and its expression
finally localizes to the rostral half of each somite
(Dunwoodie et al., 1997). The
Pudgy mutant (Dll3pu/pu, Dll3-null) embryo
exhibits expression of both rostral and caudal half marker genes, but the
patterns are spatially disorganized
(Kusumi et al., 1998
). Thus,
we cannot readily conclude from the pudgy phenotype alone whether the
Dll3-Notch signal results in activation or suppression of Dll1. To
explore the roles of Dll3 in formation of the rostrocaudal polarity of
somites, we first examined the mutual regulation of Dll3 and
Mesp2. Pudgy is a frame-shift mutation caused by a four-nucleotide
deletion (Kusumi et al.,
1998
), allowing us to analyze expression of Dll3
transcript in the Dll3pu/pu embryo. Comparison between
wild and Dll3pu/pu embryos has revealed that the rostral
stripes of Dll3 expression are lost in the absence of functional Dll3
(Fig. 3A,B)
(Kusumi et al., 1998
),
indicating that Dll3 is required for formation of the stripe pattern of its
own expression. A relatively clear boundary in the expression level was
observed between the PSM and somite region in the
Dll3pu/pu embryo. The level of Mesp2 expression
is significantly decreased in the Dll3pu/pu embryo,
suggesting that Dll3 upregulates expression of Mesp2
(Fig. 3C,D). Finally, in the
Mesp2-null embryo, instead of stripe formation, a weak diffuse Dll3
expression is expanded rostrally (Fig.
3E,F). The above observations show that Dll3 induces expression of
Dll3 itself and Mesp2, while Mesp2 suppresses expression of
Dll3. Thus, the regulatory interactions between Dll3 and
Mesp2 appear similar to those of Dll1 and Mesp2.
However, the expansion of Dll3 expression in the absence of Mesp2 is
also observed in the Dll3/Mesp2 double-null embryo, indicating that it does
not depend on Dll3 (Fig. 3G-J).
This situation is different from that for Dll1 and Mesp2
(Fig. 1Q). Thus, the regulatory
relationship between Dll3 and Mesp2 is similar to but
different from that between Dll1 and Mesp2. Taken together,
both Dll3 and Mesp2 are necessary for their mutual normal
expression. This indicates that stripe pattern of Dll3, as well as
that of Dll1, is formed by involvement of Mesp2, and not simply by
the molecular clock oscillating in the posterior PSM.
|
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Dll3-Notch signals are also both Psen1-dependent and
Psen1-independent
The expression level of Mesp2 was moderately decreased in the
Dll3-null, Psen1-null and Dll3/Psen1 double-null embryos, and they were
comparable among the three genotypes, suggesting that Mesp2
expression is partly dependent on Psen1-dependent Dll3-Notch signaling
(Fig. 5M-P). However, the
remaining Mesp2 expression observed in Dll3/Psen1 double-null embryo
is dependent on neither Dll3 nor Psen1, confirming that this expression of
Mesp2 is induced via Psen1-independent Dll1-Notch signaling as
already suggested (Fig. 5).
The expression patterns of caudal marker genes were correlated with the
morphology of the vertebrae (Fig.
6). In the Psen1-null embryo
(Dll3+/+Psen1-/-), stripes of
Dll1 and Uncx4.1 expression were completely lost, and the
pedicles of the neural arches were missing
(Fig. 6C,H,M)
(Takahashi et al., 2000).
Although blurred Dll1 expression was not detected, weak disorganized
Uncx4.1 expression was observed in the Dll3/Psen1 double-null embryo
(Dll3pu/puPsen1-/-,
Fig. 6D,I). This level of
Uncx4.1 expression was lower than that in the
Dll3pu/pu, but distinguishable from that in the
Dll3+/+Psen1-/-. This suggests that
Dll3 can suppress expression of Dll1 and Uncx4.1 in the
absence of Psen1, and Psen1 can mediate the Dll1-Notch signal to induce
expression of Dll1 and Uncx4.1 in the absence of Dll3. These
are further confirmed by the analyses of skeletal phenotypes. The vertebrae of
Dll3pu/puPsen1-/- exhibited an intermediate
morphology between Dll3pu/pu and
Dll3+/+Psen1-/- vertebrae. Whereas the
Dll3pu/pu vertebrae had a considerable amount of
disorganized skeletal elements in the position of the pedicles
(Fig. 6L), the amount of
disorganized skeletal elements was smaller in the vertebrae of
Dll3pu/puPsen1-/-
(Fig. 6N). Thus, the phenotype
of Dll3pu/puPsen1-/- embryos differs from the
phenotypes of both Dll3pu/pu and
Dll3+/+Psen1-/- embryos.
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DISCUSSION |
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There is another example that the oscillation in the posterior PSM seems to
be separated from the stripe formation. Holley et al.
(Holley et al., 2002) have
reported the interesting observation that in zebrafish embryos injected with
her1-MO, a normal stripe of deltaC expression is formed in the
anterior PSM, in the absence of oscillation of deltaC or
her1 in the posterior PSM. In this case, the deltaC stripe
at the anterior PSM is not a result of simple stabilization of oscillating
expression in the posterior PSM, but is likely to be formed by another
mechanism. This stripe formation also appears to be mediated by Notch
signaling, because the additional loss of DeltaD function disrupts stripe
formation. In addition, injection of her1/her7 double-MO completely abolishes
stripe formation (Oates and Ho,
2002
). Holley et al. suggested that Notch signaling acts in
oscillation of cyclic gene expression in the posterior PSM as well as in
stripe formation (refinement of the stripe) at the anterior PSM. We propose
that the narrowing stripe is formed at the anterior PSM, by the positive and
negative feedback loops among Dll1, Dll3 and Mesp2. These
feedback loops may constitute a kind of cellular oscillator in the anterior
PSM, which is distinct from the oscillator in the posterior PSM
(Fig. 7B). This process may be
normally linked with the oscillation process in the posterior PSM.
Interpretation of the salt-and-pepper pattern and possible functions
of Dll3
The remarkably randomized and chaotic nature of vertebrae in the pudgy
mouse has long been a mystery for geneticists. The salt-and-pepper pattern of
gene expression in the Dll3-null mouse embryo is similar to that in zebrafish
mutants aei, des and bea. Jiang et al.
(Jiang et al., 2000)
attributed this salt-and-pepper pattern to a desynchronized oscillator
activity in individual cells, and concluded that Notch signaling is not
essential for the oscillator activity itself, as the salt-and-pepper pattern
is regarded as a result of a complete lack of Notch function in zebrafish
mutants. However, we have demonstrated by genetic analysis that both
Dll1-Notch signaling via Psen1 (Fig.
6) and Mesp2 (Fig.
4) are functioning in the Dll3-null embryo
(Fig. 7B). We propose a model
for rostrocaudal patterning, where the positive and negative feedback loops of
Dll1 and Mesp2 and their integration by Dll3 are essential
(Fig. 7B). Even in the absence
of Dll3, Dll1 and Mesp2 are still expressed at considerable
levels, and interactions among adjacent cells can result in two different
states. The Dll1-Notch signal activates expression of Dll1 in
neighboring cells, thus causing upregulation of Dll1 in a group of
cells. Subsequently, the reciprocal Dll1-Notch signal also induces
Mesp2 expression, which suppresses Dll1 expression so that
Dll1 is downregulated in the cell population. When Dll1 is
downregulated, Mesp2 levels are also reduced by the lack of the
juxtacrine Dll1 signal. Thus, the cells can `oscillate' between the two states
in the absence of Dll3. With some impact of initial stochastic activation,
these interactions may produce and maintain uneven salt-and-pepper patterns of
gene expression. In the wild-type embryo, involvement of Dll3 leads to
synchronization of Dll1-dominant and Mesp2-dominant cells, and thus
integration of the stripe pattern. As Mesp2 functions to activate rostral
properties and suppresses caudal properties, the Mesp2-dominant domain is
referred to as the presumptive rostral domain. The current model is a further
development of our previous model
(Takahashi et al., 2000
). In
our previous paper we showed that the stripe of Dll1 expression is
not a remainder of strong expression in the posterior PSM, but is newly
induced via Psen1-dependent Notch signaling. That is, all the cells spanning
the future one somite region undergo suppression by Mesp2, and the
Dll1 stripe is formed after or simultaneously with this suppression.
We now interpret this process to be a result of the integration of cellular
oscillation at the individual cellular level.
What then is the synchronizing function of Dll3 at the cellular level? The salt-and-pepper pattern of Dll1 and Uncx4.1 expression in the Dll3pu/pu embryo has somewhat confused the issue of whether the Dll3-Notch signal activates or suppresses Dll1 expression. As the level of blurred and mislocalized Dll1 expression in the Dll3pu/pu embryo is lower than that of definite Dll1 stripes in the wild-type embryo, one might consider that Dll3 function is required for activation of Dll1. However, strong expansion of Dll1 expression is evident in the Dll3/Mesp2 double-null embryo, as well as in the Mesp2-null embryo, indicating that Dll3 is not necessary for the auto-activation of Dll1 via a positive feedback loop. Although the precise mechanism leading to the synchronization is yet to be defined, the likely function of Dll3 is to suppress Dll1-Notch signaling, probably in cooperation with Mesp2. This function seems feasible when considered in relation to their normal expression patterns, as the expression of Dll3 and Mesp2 finally localizes to the rostral half. Actually, the restoration of the stripe pattern of Dll1 and Uncx4.1 in the Dll3+/puPsen1-/- embryo implies that Dll3-Notch signaling can counteract Psen1-dependent Dll1-Notch signaling. This phenomenon also suggests that the stripe pattern is formed by a balance of two opposing signals. Probably, the requirement of Psen1 for the activation of Dll1 is not absolute, and in the Psen1-null embryo, a severely reduced, weak ability for Dll1 activation is overcome by suppression by Dll3-Notch signaling. Thus, reduction of the amount of the Dll3-Notch signal may restore the balance of the counteracting signals.
In the posterior PSM, Dll1 and Dll3 have essential roles in formation of
traveling waves of cyclic genes such as lunatic fringe and Hes1
(del Barco Barrantes et al.,
1999; Jouve et al.,
2000
; Dunwoodie et al.,
2002
). Therefore, we cannot exclude the possibility that Dll1 and
Dll3 influence the rostrocaudal patterning through their effects on the
molecular clock in the posterior PSM. Analysis of the possible linkage between
stripe formation at the anterior PSM and the oscillation process in the
posterior PSM is of special importance for understanding the roles of Notch
signaling in somitogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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