|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 30 January 2008
doi: 10.1242/dev.006742
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Molecular Genetics, The Ohio State University, 984 Biological Sciences Building, 484 West 12th Avenue, Columbus, OH 43210-1292, USA.
Author for correspondence (e-mail:
cole.354{at}osu.edu)
Accepted 20 December 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
FCE1). In the absence of
oscillatory Lfng expression, Notch activation is ubiquitous in the
PSM of LfngFCE1 embryos.
LfngFCE1 mice exhibit severe segmentation
phenotypes in the thoracic and lumbar skeleton. However, the sacral and tail
vertebrae are only minimally affected in LfngFCE1
mice, suggesting that oscillatory Lfng expression and cyclic Notch
activation are important in the segmentation of the thoracic and lumbar axial
skeleton (primary body formation), but are largely dispensable for the
development of sacral and tail vertebrae (secondary body formation).
Furthermore, we find that the loss of cyclic Lfng has distinct
effects on the expression of other clock genes during these two stages of
development. Finally, we find that LfngFCE1 embryos
undergo relatively normal R/C somite patterning, confirming that Lfng
roles in the segmentation clock are distinct from its functions in somite
patterning. These results suggest that the segmentation clock may employ
varied regulatory mechanisms during distinct stages of anterior/posterior axis
development, and uncover previously unappreciated connections between the
segmentation clock, and the processes of primary and secondary body
formation.
Key words: Lunatic fringe, Notch, Segmentation clock, Somitogenesis, Secondary body formation, Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Gossler and Hrabe de
Angelis, 1998). This process is dynamic and complex. During
gastrulation, cells enter the presomitic mesoderm via the primitive streak.
Later in development (
10.0 dpc) the tailbud forms, and further mesodermal
cells arise from this structure (Gossler
and Tam, 2002). Although this distinction between primary body
formation (giving rise to cervical, thoracic and lumbar vertebrae) and
secondary body formation (giving rise to post-anal structures) was originally
proposed in 1925 (Holmdahl,
1925), it remains unclear how, and to what extent, the genetic
regulation of somitogenesis between these two processes may vary (reviewed by
Handrigan, 2003).
Several models for the control of somitogenesis invoke a clock that
provides a timing mechanism for segmentation
(Cooke and Zeeman, 1976;
Kerszberg and Wolpert, 2000;
Meinhardt, 1986;
Schnell and Maini, 2000).
These models differ in their specifics, but all include an oscillating
activity in the PSM with a period identical to the rate of somite formation.
Molecular evidence for the segmentation clock came initially from the cyclic
expression pattern of chicken c-hairy RNA
(Palmeirim et al., 1997).
Shortly thereafter, lunatic fringe (Lfng) RNA was found to have
oscillatory expression patterns in the PSM, linking it to the clock as well
(Aulehla and Johnson, 1999;
Forsberg et al., 1998;
McGrew et al., 1998).
The importance of Notch signaling during vertebrate segmentation is evident
from the phenotypes associated with mutations in Notch pathway genes, many of
which cause defects in embryonic segmentation. Furthermore, cyclic gene
expression has been described in the presomitic mesoderm for many other genes
linked to the Notch signaling pathway in mouse, zebrafish and chick (reviewed
by Rida et al., 2004;
Shifley and Cole, 2007). The
Wnt pathway has also been linked to the clock. Both Axin2 and
Nkd1 RNA levels oscillate in the PSM, and it has been suggested that
the Wnt pathway lies upstream of oscillatory Notch signaling
(Aulehla et al., 2003;
Ishikawa et al., 2004). More
recently, a large number of oscillatory genes have been identified, many of
which are linked to the Notch, Wnt or FGF pathways
(Dequeant et al., 2006),
suggesting complex clock regulation involving multiple signaling pathways.
The analysis of Notch signaling in the segmentation clock mechanism is
complicated by the fact that this pathway plays multiple roles during
somitogenesis. The PSM can be divided into functionally distinct regions based
on RNA expression patterns (reviewed by
Saga and Takeda, 2001). In the
posterior PSM (region I), cyclic expression of several genes reflects the
function of the segmentation clock. In the anterior PSM (region II) the
expression of the cycling genes is stabilized, and the pre-somites develop
rostral and caudal compartments. These regions are demarcated by the graded
expression of FGF8 in region I, which has been suggested to maintain the
immature state of the cells (Dubrulle et
al., 2001; Sawada et al.,
2001). Several lines of evidence suggest that Notch signaling
plays distinct roles in these two regions. In region I of the PSM, Notch
activity levels oscillate, suggesting its function in this region is linked to
the clock (Huppert et al.,
2005; Morimoto et al.,
2005). Some models suggest that this oscillatory activation may be
achieved partially through the transitory inhibition of Notch signaling via
its glycosylation by LFNG in the Golgi, and transcriptional feedback loops
involving Hes7 (Dale et al.,
2003). This oscillatory mechanism, however, clearly receives input
from other members of the Notch pathway and from other signaling pathways,
including Wnt and FGF. This complex network of interlocked oscillatory genes
has been proposed to contribute to the robust nature of somitogenesis
(Dequeant et al., 2006). Notch
signaling also plays crucial roles in the patterning of the presumptive
somites in region II of the PSM. It appears that interplay between the Mesp
genes and the Notch pathway is required for the establishment of rostrocaudal
polarity in the developing somites, with Mesp2 acting through the
Notch pathway to downregulate Dll1 expression in the presumptive
rostral somite compartment, while in the presumptive caudal compartment, Notch
signaling upregulates Dll1 expression. Lfng is a direct
target of Mesp2, and its stable expression in the rostral compartment
may inhibit Notch signaling in this compartment
(Morimoto et al., 2005;
Takahashi et al., 2000).
We and others have defined genomic sequences sufficient to direct cyclic
expression of Lfng in the PSM, and demonstrated that independent
Lfng cis-acting regulatory regions drive stable RNA expression in the
rostral compartment of the developing somites in the anterior PSM
(Cole et al., 2002;
Morales et al., 2002).
Deletion of a conserved regulatory element termed fringe clock element 1
(FCE1) from Lfng reporter transgenes eliminates cyclic expression in
the caudal PSM, while maintaining expression in the anterior PSM, reflecting
the distinct roles of Lfng in the segmentation clock and in R/C
patterning of developing somites. Thus, the complex phenotypes of
Lfng-/- mice may arise from disruption of both of these
roles, with variations in somite size perhaps resulting from impaired clock
function, while the apparent mingling of somite compartments might be
exacerbated by altered R/C patterning.
To dissect the functions of the Notch pathway during segmentation, we perturbed only one of the roles of Notch signaling, by disrupting oscillatory Lfng expression in region I of the PSM, while sparing its expression in region II of the PSM. We report here that the clock and patterning roles of Lfng during somitogenesis are functionally separable. Strikingly, we find that the loss of oscillatory Lfng expression and Notch1 activity in region I of the PSM has more severe effects during the segmentation of the thoracic and lumbar skeleton than the sacral and tail skeleton. This suggests that oscillatory Notch1 activation in the segmentation clock is much more important during primary body formation than during secondary body formation. By contrast, the specific localization of Notch activity to the presumptive caudal compartment of the pre-somite in region II of the PSM is important throughout development.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
).
3' flanking sequences extended from the HindIII site to the
XhoI site in intron 1. Linearized vector was electroporated into TC1
cells (Deng et al., 1996) and
G418 resistant colonies were screened by Southern blot. Two independent ES
cell lines were injected by the OSUCCC Transgenic/ES Core Facility, and
transmitted through the germline. Results from the lines were identical and
are combined. Mice were maintained on a mixed 129/SvxC57BL/6J
background. LfngtmRjo1/+ mice (R. Johnson), were
maintained on a mixed 129/SvxC57BL/6J background, or crossed one
generation with FVB/J mice to increase the recovery of adult
LfngtmRjo1/tmRjo1 mice (referred to as
Lfng-/-). Mice were maintained under the care of the Ohio
State University ILACUC.
Genotyping
Genomic DNA was prepared from tail clips via proteinase K saltout or from
yolk sac fragments via the HOTSHOT procedure
(Truett et al., 2000). Animals
were genotyped by PCR. LfngtmRjo1 primers FNG322
(5'-GAGCACCAGGAGACAAGCC-3'), FNG325
(5'-AGAGTTCCTGAAGCGAGAG-3') and PGK3
(5'-CTTGTGTAGCGCCAAGTGC-3') amplify a 170 bp wild-type product and
a 200 bp mutant product. LfngFCE1 primers SC284
(5'-TTTGGTGGGAATGGATTAGC-3') and SC285
(5'-CTGGTCCATTTGCTCTGAGG-3') produce a 340 bp wild-type and a 182
bp mutant bands, while SC286 (5'-TTGGGTCTATCTGGGAAACG-3') and
SC287 (5'-GCGACTCATCCAGACACAGA-3') produce a 149 bp wild-type and
a 250 bp mutant bands.
Whole mount in situ hybridization
Embryos were collected from timed pregnancies (noon of the day of plug
identification designated as 0.5 dpc). RNA in situ hybridization using
digoxigenin-labeled probes was performed essentially as described
(Riddle et al., 1993);
however, embryos were blocked in a mixture of MABT +20% sheep serum +2%
Boehringer blocking reagent, and all post-antibody washes were performed in
MABT. Hes7 cDNA probes extend from the internal SmaI site to
the stop codon. Hes7 intron probe was amplified using the primers
5'-GCTAGAGGCCATAGCTGGTG and 5'-CTGTGACCAGCGGGAAAG. Dll1
intron probe was amplified using primers 5'-GTTGGCAGTGGGAAGAAGG and
5'-TGTGTTGTGCCAATGAAGGT. Nrarp probe was amplified using the
primers 5'-GCGTGGTTATGGGAGAAAGA and 5'-TTCCTCCCACACTGGTTCAT. The
Hesr1 probe comprises the coding region of the cDNA. Other probes
were Lfng (Johnston et al.,
1997), Mesp2 (Saga et
al., 1997), Uncx4.1
(Mansouri et al., 1997) and
Mox2 (Candia et al.,
1992).
Skeletal preparations, neurofilament staining and histology
Skeletal preparations of neonates or 18.5 dpc embryos were performed
essentially as described (Kessel and
Gruss, 1991). Neurofilament staining was performed using the 2H3
antibody (Developmental Studies Hybridoma Bank), using standard protocols. For
histological analysis, embryos were fixed in Bouin's fixative and transferred
to ethanol for storage. Embryos were embedded in paraffin and 10 µm
sections were stained with Haemotoxylin and Eosin.
Whole mount immunohistochemistry
Embryos were fixed in fresh 4% PFA in PBS, then washed in PBS. After
overnight incubation at 4°C in PBS containing 0.1% hydrogen peroxide, 1%
Triton X-100 and 10% fetal calf serum (TS-PBS), embryos were transferred into
10 mM sodium citrate (pH 6.0), 0.1% Tween-20 (CT), boiled for 10 minutes and
then transferred back to PBS. After washing in TS-PBS, embryos were incubated
for 5 days in primary Cleaved Notch1 (Val1744) antibody (Cell Signaling
Technology) in TS-PBS (1:250). After washing embryos were incubated overnight
in AP-conjugated secondary antibody in MABT (1:500). After washing, embryos
were transferred to NTMT and stained with BCIP/NBT as described
(Riddle et al., 1993).
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
FCE1 (Fig.
1A,B). We hypothesized that this mutation would disrupt expression
of Lfng in the caudal PSM (region I), where the clock is active,
while preserving Lfng expression in the anterior PSM (region II),
where R/C somite patterning is initiated.
Lfng expression is perturbed in the PSM of
LfngFCE1/FCE1 mutant embryos. In wild-type
embryos, three distinct phases of Lfng expression are seen in the
PSM, reflecting cyclic expression (Fig.
1C, parts c-f). By contrast,
LfngFCE1/FCE1 embryos express Lfng
RNA in a single band in the anterior PSM, with no expression observed in the
caudal PSM where the clock is active (Fig.
1C, parts g,h). Similar results were seen at stages between 8.5
and 11.5 dpc (data not shown). Although the anterior band of Lfng
expression in LfngFCE1/FCE1 embryos is
weaker than the anterior-most band of Lfng expression in wild-type
embryos, these results demonstrate that the deletion of the FCE1 enhancer
prevents oscillatory expression of Lfng in region I of the PSM, while
sparing some level of expression in region II. In addition, we find that
Lfng expression in region II of the PSM is largely confined to the
presumptive rostral compartment of somite S-1 (data not shown), indicating
that the endogenous Lfng expression pattern in the anterior PSM is
preserved in the LfngFCE1 allele.
|
FCE1/FCE1 animals survive to
adulthood at Mendelian ratios, and homozygous animals of both sexes are
fertile. LfngFCE1/FCE1 animals have
segmentation defects, including shortened body and variably kinked tails
(Fig. 2A). In the anterior
skeleton, both Lfng-/- and
LfngFCE1/FCE1 animals are severely affected.
Multiple rib fusions and bifurcations as well as severely disorganized
vertebrae are observed (Fig.
2B). When defects in the thoracic region of the skeleton are
quantified, we find similar levels of disorganization in
LfngFCE1/FCE1 and
Lfng-/- animals (Fig.
2C).
In the more posterior skeleton, however,
LfngFCE1/FCE1 animals are much less affected
than Lfng-/- animals
(Fig. 2B,D). In the thoracic
and lumbar region of the skeleton, vertebral condensations in both
LfngFCE1/FCE1 and
Lfng-/- animals are irregular and misaligned. Strikingly,
this pattern is altered at the lumbo-sacral junction. In the sacral region of
LfngFCE1/FCE1 animals, normal vertebral
condensations are seen in all animals, and the tail vertebrae appear
relatively normal, though variable kinks in the tail are seen ranging from
mild (0-1 in 40% of mice) to moderate (2-5 in 60% of mice). By contrast, in
Lfng-/- animals, vertebral condensations are abnormal
throughout the sacral region and the tail appears truncated, a phenotype never
seen in LfngFCE1/FCE1 animals
(Fig. 2B,D). Thus we find that
the loss of oscillatory Lfng expression in region I of the PSM causes
pronounced defects in the axial skeleton, but these defects are much more
pronounced in the thoracic and lumbar regions, while the sacral and more
caudal regions of the skeleton are less affected in comparison to the null
allele. Interestingly, the lumbo-sacral junction, the point where skeletal
morphology largely recovers in LfngFCE1/FCE1
animals, represents the transition point between primary and secondary body
formation, suggesting that oscillatory Lfng plays, at most, a minor
role in secondary body formation.
The LfngFCE1 allele affects somite formation differently during primary and secondary body formation
To test the hypothesis that early and late somitogenesis are differentially
affected by the loss of oscillatory Lfng expression, we examined somite
morphology at different stages of embryonic development. Somites that
contribute to the thoracic and lumbar regions of the skeleton are produced
during primary body formation (Gossler and
Tam, 2002). In LfngFCE1/FCE1
embryos, these somites are irregularly sized and spaced with frequent fusions
between neighboring somites (Fig.
3B), and the mature derivatives of these somites remain
irregularly spaced and sized at 10.5 dpc
(Fig. 3D). During secondary
body formation, however, somite development recovers in
LfngFCE1/FCE1 embryos, producing relatively evenly
sized and spaced epithelial somites (Fig.
3F). These data support the idea that the loss of oscillatory
Lfng expression differentially affects primary and secondary body
formation with thoracic and lumbar somites being more sensitive to the loss of
cyclic Lfng activity than are more caudal somites.
Rostral-caudal somite patterning is partially rescued in LfngFCE1/FCE1 embryos
Lfng-/- embryos have severe defects in R/C somite
patterning (Evrard et al.,
1998; Zhang and Gridley,
1998). To address whether the Lfng expression in region
II of the PSM of LfngFCE1/FCE1 embryos could
rescue R/C patterning, we examined compartment formation in the anterior PSM
and in mature somites of Lfng mutant mice. We examined R/C patterning
in region II of the PSM by assessing the expression of Mesp2. Mesp2
defines the presumptive rostral compartment of somite S-1, and interacts with
Lfng and Notch1 signaling during the process of R/C patterning
(Morimoto et al., 2005;
Takahashi et al., 2000).
During both primary and secondary body formation, we find that Mesp2
is expressed in a single band of varying width in the anterior PSM of both
wild-type and LfngFCE1/FCE1 embryos,
reflecting the early expression and subsequent refinement of Mesp2 in
the presumptive rostral compartment. However, in
LfngFCE1/FCE1 embryos, we frequently see a
less distinct rostral border, regardless of the stage of somitogenesis
(Fig. 4A). These results
demonstrate that the rostral compartment is being defined in the presomites in
region II of LfngFCE1/FCE1 embryos
throughout somitogenesis but may suggest that this earliest marker of
patterning is mildly disrupted. As this disruption is seen throughout
somitogenesis, it may be due to the reduced dose of Lfng in the
anterior PSM, rather than to differences in primary and secondary body
formation.
|
;
Zhang and Gridley, 1998). In
LfngFCE1/FCE1 embryos, Uncx4.1
expression in newly formed somites is largely compartmentalized, with stronger
expression in the more caudal region of somites S1 and S2. Clearer
compartmentalization is observed in more anterior somites, but compartments
are frequently irregularly spaced (Fig.
4B, parts e,f). Compartmentalization of mature somites in the
thoracic region of LfngFCE1/FCE1 embryos is
more distinct by 10.5 dpc, with clear bands of Uncx4.1 visible in the
sclerotome. Again, compartments of Uncx4.1 expression are frequently
irregularly spaced or shaped, presumably reflecting the irregularities in
somite size and shape observed morphologically in the thoracic region of the
embryo (Fig. 4B, part g).
Similar results are seen when examining Mox1 at 10.5 dpc
(Fig. 4C). By contrast, the
Uncx4.1 signal in the thoracic region of Lfng-/-
embryos fails to compartmentalize, maintaining an unsegmented pattern
(Fig. 4B, part k). The somites
formed during secondary body formation in
LfngFCE1/FCE1 embryos are clearly
compartmentalized with regular rostral and caudal segmentation, whereas in
Lfng-/- embryos, R/C patterning continues to be abnormal
(Fig. 4B, parts h,l).
Functionality of R/C patterning was assessed by neurofilament staining with
2H3. In wild-type embryos, regular neurofilament staining is observed,
representing the axonal trajectories of spinal neurons through the rostral
somite compartment. In LfngFCE1/FCE1
embryos, axonal projections are seen, but their spacing is irregular
(Fig. 4D). Thus, although
LfngFCE1/FCE1 embryos produce irregular
somites during primary body formation, the retention of Lfng
expression in the anterior PSM supports relatively normal R/C patterning, and
somites formed during secondary body formation undergo normal R/C patterning.
This supports the idea that the role of Lfng in R/C somite patterning
is distinct and separable from its functions in the segmentation clock.
|
FCE1 allele on Notch1 signaling, we visualized
Notch activation using an antibody specific for the Notch1 ICD (NICD). Notch
signaling levels oscillate in the PSM of wild-type embryos during primary and
secondary body formation (Fig.
5) (Morimoto et al.,
2005), with different patterns of NICD staining found in different
embryos. By contrast, in both LfngFCE1/FCE1
(Fig. 5) and
Lfng-/- (Fig.
5) (Morimoto et al.,
2005) embryos, a gradient of NICD is seen in the PSM, reflecting
ubiquitous, non-oscillatory Notch signaling throughout the PSM. This confirms
that the LfngFCE1 allele inhibits oscillatory Notch
signaling in region I of the PSM during both primary and secondary body
formation, and indicates that oscillatory Notch activation in region I of the
PSM is largely dispensable for segmentation during secondary body
formation.
Expression of oscillatory genes is differentially affected during primary and secondary body formation in LfngFCE1/FCE1 embryos
To assess the effects of the loss of cyclic Lfng expression on the
transcription of other segmentation clock genes, we first examined the
expression of Hes7 in LfngFCE1/FCE1
embryos. Hes7 has been proposed to play a role in the segmentation
clock mechanism as part of the feedback loops regulating oscillatory
Lfng transcription and Notch1 activation (reviewed by
Rida et al., 2004). One report
has suggested that Hes7 expression is ubiquitous in the
Lfngtm1Grid/tm1Grid null background at 9.5 dpc
(Chen et al., 2005), while more
recent results suggest the Hes7 expression is affected but still
dynamic in the absence of Lfng
(Niwa et al., 2007). During
primary body formation, we used a probe specific for Hes7 intronic
sequences to show that Hes7 RNA is transcribed in a stable ubiquitous
pattern in the PSMs of LfngFCE1/FCE1 and
Lfng-/- embryos, distinct from the dynamic banding pattern
seen in wild-type embryos (Fig.
6A). Thus, during primary body formation, the loss of
Lfng prevents the cyclic transcription of Hes7. In sharp
contrast, we find that during secondary body formation, Hes7
transcription oscillates in the same way as wild-type expression patterns in
both LfngFCE1/FCE1 and
Lfng-/- embryos (Fig.
6B). Similar results were seen using a Hes7 mRNA probe,
indicating that post-transcriptional regulation of Hes7 mRNA levels
is also normal in these embryos (Fig.
6C). Hes7 cyclic expression was confirmed by half tail
culture experiments. PSMs were bisected, with one half fixed immediately and
the other half cultured before fixation. After 1 hour of culture, the
Hes7 expression pattern in the cultured half is different from the
uncultured half regardless of genotype
(Fig. 6D). These results
suggest that Hes7 transcription may be differentially controlled
during different stages of somitogenesis, requiring Notch oscillations during
primary body formation, but not during secondary body formation.
We confirmed and extended these observations by analyzing the expression of
other oscillatory genes in LfngFCE1/FCE1
embryos. Similar to our results with Hes7, we find that
Nrarp expression is differentially affected during primary and
secondary body formation. Before tailbud formation, distinctive banding
patterns are observed in wild-type, but not in
LfngFCE1/FCE1 embryos
(Fig. 7A). After tailbud
formation, oscillatory Nrarp expression recovers in
LfngFCE1/FCE1 embryos, although the cyclic
expression patterns are less distinct than those in wild-type embryos. Other
Notch pathway genes also oscillate during secondary body formation in
LfngFCE1/FCE1 embryos, including
Dll1 (Fig. 7B), but
the expression of some Notch targets, including Hesr1 is perturbed at
this stage (Fig. 7C). Thus,
although multiple genes that may be involved in the segmentation clock
mechanism exhibit oscillatory expression in the absence of cyclic Notch
activation during secondary body formation in
LfngFCE1/FCE1 embryos, cyclic Notch1
activity is required for proper expression of some genes in the region at this
stage.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). We
therefore specifically disrupted the oscillatory expression of Lfng
in region I of the PSM to examine the role it plays in the segmentation clock
during mouse somitogenesis. Targeted deletion of FCE1 sequences eliminates
expression of Lfng in the posterior region of the PSM, indicating
that the enhancer is required for cyclic Lfng transcription in region
I of the PSM. However, the anterior band of Lfng expression in
LfngFCE1/FCE1 embryos is weaker than that
seen in wild-type embryos, perhaps supporting an additional role for FCE1 in
enhancing expression of Lfng in the anterior PSM.
|
FCE1/FCE1 embryos, but distinct effects
are seen during primary and secondary body formation. In the thoracic and
lumbar skeleton, malformed vertebral condensations and rib abnormalities were
seen in LfngFCE1/FCE1 skeletons
(Fig. 2B). The appearance of
the vertebrae resembles the phenotypes seen in some cases of autosomal
recessive spondylocostal dysostosis caused by mutations in DLL3 or
LFNG; both the thoracic and lumbar spine are affected and vertebral
bodies are irregularly shaped and fitted together
(Bulman et al., 2000;
Sparrow et al., 2006). To our
surprise, we found that the caudal skeletal regions (sacral and tail
vertebrae) were invariably less severely affected in
LfngFCE1/FCE1 animals than in
Lfng-/- animals. Especially striking is the fact that in
the sacral region of the spine,
LfngFCE1/FCE1 animals exhibit essentially
normal vertebral formation, whereas irregularities are still seen at this
level in Lfng-/- skeletons. The point of phenotype
recovery at the lumbo-sacral junction indicates that secondary body formation
occurs relatively normally in LfngFCE1/FCE1
embryos.
This differential severity is reflected in the process of somitogenesis
throughout development. During primary body formation, somites are frequently
abnormal in LfngFCE1/FCE1 embryos
(Fig. 3). The production of
irregularly sized somites suggests that the loss of oscillatory Lfng
expression in region I of the PSM interferes with segmentation clock function
during primary body formation. By contrast, during secondary body formation,
somites formed in LfngFCE1/FCE1 embryos are
evenly spaced and of regular size, and the phenotypes in the sacral and caudal
skeleton are correspondingly milder (Figs
2 and
3). Thus, our data suggest that
segmentation of the embryo during primary body formation (contributing to the
thoracic and lumbar skeleton) is more sensitive to the loss of cyclic
Lfng expression than is segmentation during secondary body formation.
This sheds new light on one of the classical issues of developmental biology:
the extent to which primary and secondary body formation represent distinct
mechanisms of development.
Dll3-null embryos exhibit similar Lfng expression
patterns to those observed in LfngFCE1/FCE1
mice, with expression observed only in the anterior PSM after 9.5 dpc
(Dunwoodie et al., 2002;
Kusumi et al., 2004).
Interestingly, Dll3-null mice exhibit disordered somitogenesis along
the length of the vertebral column, suggesting that Lfng expression
in region II of the PSM is not, in and of itself, sufficient to rescue
secondary body formation. This may reflect a requirement for Dll3
expression in the anterior PSM during secondary body formation. Alternatively,
it was recently shown that the loss of Dll3 in the PSM leads to a
loss or reduction in NICD levels in region I of the PSM, in contrast to the
ubiquitous Notch1 activation observed in
LfngFCE1/FCE1 embryos
(Geffers et al., 2007). This
raises the possibility that while oscillatory Notch1 activation in the
posterior PSM is not required during secondary body formation, some level of
Notch1 activation is still necessary during this process. This may be
especially interesting in light of the observation that constitutive
overexpression of Lfng in the mouse PSM, which might be predicted to
repress Notch1 activation, also perturbs somitogenesis along the entire axial
skeleton (Serth et al.,
2003).
|
; Zhang and Gridley,
1998). This could arise due to downstream effects of the loss of
Lfng in the segmentation clock, or more directly due to the loss of
Lfng expression in region II of the PSM. Analysis of R/C patterning
in LfngFCE1/FCE1 embryos directly assesses
whether the retention of Lfng expression in the presumptive anterior
compartment of the forming somite can rescue R/C patterning in the absence of
oscillatory Notch activity in the clock. During secondary body formation,
LfngFCE1/FCE1 embryos produce regular pairs
of somites, and these somites are properly patterned. More surprisingly, the
irregular somites produced during primary body formation in
LfngFCE1/FCE1 embryos are also patterned
into clear rostral and caudal compartments
(Fig. 4B), although this
patterning may be somewhat delayed. We propose that in
LfngFCE1/FCE1 embryos, Mesp2
expression in the anterior compartment of the developing somite is able to
stabilize the pattern of Notch activation in somites S0 and S-1, at least in
part via its specific activation of Lfng transcription. This allows
the Notch pathway to function in R/C patterning in
LfngFCE1/FCE1 embryos despite the loss of
cyclic Lfng expression in region I of the PSM. It is not clear at
this time whether the delay in robust R/C patterning of thoracic somites is
due to some underlying disorganization of somites S-1 and S0 as a result of
perturbed clock function, or whether it may be a result of the reduced
Lfng levels seen in the anterior PSM in
LfngFCE1/FCE1 embryos. However, the
successful patterning of irregularly sized somites during primary body
formation in LfngFCE1/FCE1 embryos suggests
that the Notch-based processes involved in the segmentation clock can be
largely divorced from its roles in R/C somite patterning, and that the
processes regulated by oscillatory Notch signaling in region I of the PSM are
not prerequisites for the patterning of the pre-somites in region II.
Differential segmentation clock regulation at distinct levels of the axial skeleton?
The loss of cyclic Notch1 activation has distinct effects during primary
and secondary body formation. During early stages of somitogenesis, the loss
of oscillatory Lfng expression interferes with oscillatory Notch
activation (Fig. 5A) and causes
phenotypes (irregular somite size and positioning, alteration of oscillatory
gene expression) that suggest defects in segmentation clock function
(Fig. 3,
Fig. 6A). By contrast, during
later stages of segmentation, despite the continued absence of oscillatory
Lfng and the presence of ubiquitous Notch1 activation, somitogenesis
proceeds relatively normally in
LfngFCE1/FCE1 embryos, and the oscillatory
expression of several clock genes largely recovers at these stages
(Fig. 6B-D,
Fig. 7). Although expression of
some Notch target genes is slightly affected during secondary body formation
in LfngFCE1/FCE1 embryos, the mild
phenotypes observed in the caudal axial skeleton suggest that these
perturbations are relatively unimportant. Thus, it appears that segmentation
clock function is more sensitive to the loss of oscillatory Lfng
expression during primary body formation than during secondary body
formation.
Differential regulation of somitogenesis at different axial levels of the
embryo is not unprecedented. The first five or six somites are frequently
spared in mutations that affect the Notch signaling pathway, although in
zebrafish these segments can be affected by the simultaneous downregulation of
several clock components (Oates et al.,
2005) perhaps indicating multiple, parallel mechanisms of
regulation. More recently, it has been shown that in zebrafish the anlagen of
the anterior trunk, posterior trunk and tail are specified before
somitogenesis begins, raising the possibility that different genetic pathways
may affect these regions in distinct ways
(Szeto and Kimelman,
2006).
|
). For example, recent findings suggest that FGF
signaling is required for oscillatory function of both the Notch and Wnt
pathways in the PSM (Wahl et al.,
2007), though most observations were confined to primary body
formation. Our data support the additional hypothesis that oscillation of any
individual pathway or component may be more or less important during different
stages of somitogenesis. Our finding that Hes7 oscillations recover
during secondary body formation is especially interesting in light of recent
findings that Hes7 oscillations are regulated in part by the FGF
pathway, and that oscillatory HES7 protein regulates the expression of FGF
pathway components (Niwa et al.,
2007). It is clear that regulated crosstalk among these pathways
is important; however, our results suggest that specific interactions may be
differentially regulated during primary and secondary body formation.
Wnt activity may play especially important roles in the regulation of
posterior somitogenesis. Reductions in Wnt signaling levels can preferentially
affect segmentation of the posterior embryo: the Wnt3avt
hypomorphic allele develops segmentation defects in the lumbar, sacral and
tail regions, and mutations in Lrp6, encoding a Wnt co-receptor,
affect the caudal axial skeleton more severely than anterior skeletal regions
(Kokubu et al., 2004;
Pinson et al., 2000). These
data may indicate that the caudal skeleton is more sensitive to perturbations
in Wnt pathway activity. Conversely, based on our data, Notch oscillations may
play a more important role during the development of the thoracic and lumbar
skeleton. It will clearly be important to carefully dissect the interactions
among these three pathways to clarify fully the possibility that the
segmentation clock mechanism is differentially regulated during primary and
secondary body formation.
R/C patterning of anterior somites may affect ongoing segmentation during secondary body formation
We find that many aspects of clock function recover in both
Lfng-/- and
LfngFCE1/FCE1 embryos after tailbud
formation; however, the posterior skeletal phenotypes of
Lfng-/- animals are much more severe than those seen in
LfngFCE1/FCE1 embryos. Therefore, we propose
that the truncation of posterior skeletal structures in
Lfng-/- animals is caused by the perturbation of R/C
patterning in these embryos, rather than the loss of oscillatory Notch
activity in the clock. Several lines of evidence suggest that R/C somite
patterning contributes to the proper segmentation of the posterior embryo.
Targeted deletion of Mesp2, which is exclusively expressed in region
II of the PSM, causes disrupted R/C somite patterning and truncation of the
posterior skeleton (Saga et al.,
1997). Furthermore, it appears that the total dosage of MESP
activity (comprising the additive effects of MESP1 and MESP2) in region II of
the PSM is important. Manipulating the levels of MESP proteins can both
partially rescue the R/C patterning of the somites and mitigate the caudal
truncation of the axial skeleton (Morimoto
et al., 2006). In addition, in newly developed Mesp2
knockout alleles, Mesp1 expression is elevated leading to partial
rescue of somitogenesis during secondary body formation
(Takahashi et al., 2007).
Interestingly, an Mesp2 mutation found in spondylocostal dysostosis
also has more severe effects on the thoracic vertebrae than the more caudal
skeleton (Whittock et al.,
2004).
|
FCE1/FCE1 embryos, permits posterior
segmentation to proceed relatively normally, preventing the tail truncation
seen in Lfng-/- animals. This underscores the potential
for the transfer of information between the anterior and posterior regions of
the PSM, at least during secondary body formation. Thus, the work reported
here uncovers new levels of complexity linking differential regulation of
clock function and R/C somite patterning to the long-known but
little-understood processes of primary and secondary body formation. |
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: The University of Toledo, Toledo, OH 43606-3390, USA
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Aulehla, A. and Johnson, R. L. (1999). Dynamic
expression of lunatic fringe suggests a link between notch signaling and an
autonomous cellular oscillator driving somite segmentation. Dev.
Biol. 207,49
-61.[CrossRef][Medline]
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.[CrossRef][Medline]
Bulman, M. P., Kusumi, K., Frayling, T. M., McKeown, C.,
Garrett, C., Lander, E. S., Krumlauf, R., Hattersley, A. T., Ellard, S. and
Turnpenny, P. D. (2000). Mutations in the human delta
homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.
Nat. Genet. 24,438
-441.[CrossRef][Medline]
Bunting, M., Bernstein, K. E., Greer, J. M., Capecchi, M. R. and
Thomas, K. R. (1999). Targeting genes for self-excision in
the germ line. Genes Dev.
13,1524
-1528.
Candia, A. F., Hu, J., Crosby, J., Lalley, P. A., Noden, D.,
Nadeau, J. H. and Wright, C. V. (1992). Mox-1 and Mox-2
define a novel homeobox gene subfamily and are differentially expressed during
early mesodermal patterning in mouse embryos.
Development 116,1123
-1136.[Abstract]
Chen, J., Kang, L. and Zhang, N. (2005).
Negative feedback loop formed by Lunatic fringe and Hes7 controls their
oscillatory expression during somitogenesis. Genesis
43,196
-204.[CrossRef][Medline]
Christ, B., Schmidt, C., Huang, R., Wilting, J. and
Brand-Saberi, B. (1998). Segmentation of the vertebrate body.
Anat. Embryol. 197,1
-8.[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.[CrossRef][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.[CrossRef][Medline]
Dale, J. K., Maroto, M., Dequeant, M. L., Malapert, P., McGrew,
M. and Pourquie, O. (2003). Periodic notch inhibition by
lunatic fringe underlies the chick segmentation clock.
Nature 421,275
-278.[CrossRef][Medline]
Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A. and Leder, P.
(1996). Fibroblast growth factor receptor 3 is a negative
regulator of bone growth. Cell
84,911
-921.[CrossRef][Medline]
Dequeant, M. L., Glynn, E., Gaudenz, K., Wahl, M., Chen, J.,
Mushegian, A. and Pourquie, O. (2006). A complex oscillating
network of signaling genes underlies the mouse segmentation clock.
Science 314,1595
-1598.
Dubrulle, J., McGrew, M. J. and Pourquie, O.
(2001). FGF signaling controls somite boundary position and
regulates segmentation clock control of spatiotemporal Hox gene activation.
Cell 106,219
-232.[CrossRef][Medline]
Dunwoodie, S. L., Clements, M., Sparrow, D. B., Sa, X., Conlon,
R. A. and Beddington, R. S. (2002). Axial skeletal defects
caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are
associated with disruption of the segmentation clock within the presomitic
mesoderm. Development
129,1795
-1806.
Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. and Johnson, R.
L. (1998). lunatic fringe is an essential mediator
of somite segmentation and patterning. Nature
394,377
-381.[CrossRef][Medline]
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.[CrossRef][Medline]
Geffers, I., Serth, K., Chapman, G., Jaekel, R.,
Schuster-Gossler, K., Cordes, R., Sparrow, D. B., Kremmer, E., Dunwoodie, S.
L., Klein, T. et al. (2007). Divergent functions and distinct
localization of the Notch ligands DLL1 and DLL3 in vivo. J. Cell
Biol. 178,465
-476.
Gossler, A. and Tam, P. P. (2002).
Somitogenesis: segmentation of the paraxial mesoderm and the delineation of
tissue compartments. In Mouse Development (ed. J.
Rossant and P. P. Tam), pp. 122-149. San Diego:
Academic Press.
Gossler, A. and Hrabe de Angelis, M. (1998).
Somitogenesis. Curr. Top Dev. Biol.
38,225
-287.[Medline]
Handrigan, G. R. (2003). Concordia discors:
duality in the origin of the vertebrate tail. J. Anat.
202,255
-267.[Medline]
Holmdahl, D. E. (1925). Experimentelle
Untersuchungen uber die Lage der Grenze zwischen primarer und sekundarere
Korperentwicklung beim Huhn. Anat. Anz.
59,393
-396.
Huppert, S. S., Ilagan, M. X., De Strooper, B. and Kopan, R.
(2005). Analysis of Notch Function in presomitic mesoderm
suggests a gamma-secretase-independent role for presenilins in somite
differentiation. Dev. Cell
8, 677-688.[CrossRef][Medline]
Ishikawa, A., Kitajima, S., Takahashi, Y., Kokubo, H., Kanno,
J., Inoue, T. and Saga, Y. (2004). Mouse Nkd1, a Wnt
antagonist, exhibits oscillatory gene expression in the PSM under the control
of Notch signaling. Mech. Dev.
121,1443
-1453.[CrossRef][Medline]
Johnston, S. H., Rauskolb, C., Wilson, R., Prabhakaran, B.,
Irvine, K. D. and Vogt, T. F. (1997). A family of mammalian
Fringe genes implicated in boundary determination and the Notch pathway.
Development 124,2245
-2254.[Abstract]
Kerszberg, M. and Wolpert, L. (2000). A clock
and trail model for somite formation, specialization and polarization.
J. Theor. Biol. 205,505
-510.[CrossRef][Medline]
Kessel, M. and Gruss, P. (1991). Homeotic
transformations of murine vertebrae and concomitant alteration of Hox codes
induced by retinoic acid. Cell
67, 89-104.[CrossRef][Medline]
Kokubu, C., Heinzmann, U., Kokubu, T., Sakai, N., Kubota, T.,
Kawai, M., Wahl, M. B., Galceran, J., Grosschedl, R., Ozono, K. et al.
(2004). Skeletal defects in ringelschwanz mutant mice reveal that
Lrp6 is required for proper somitogenesis and osteogenesis.
Development 131,5469
-5480.
Kusumi, K., Mimoto, M. S., Covello, K. L., Beddington, R. S.,
Krumlauf, R. and Dunwoodie, S. L. (2004). Dll3 pudgy mutation
differentially disrupts dynamic expression of somite genes.
Genesis 39,115
-121.[CrossRef][Medline]
Mansouri, A., Yokota, Y., Wehr, R., Copeland, N. G., Jenkins, N.
A. and Gruss, P. (1997). Paired-related murine homeobox gene
expressed in the developing sclerotome, kidney, and nervous system.
Dev. Dyn. 210,53
-65.[CrossRef][Medline]
McGrew, M. J., Dale, J. K., Fraboulet, S. and Pourquie, 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.[CrossRef][Medline]
Meinhardt, H. (1986). Models of segmentation.
In Somites in Developing Embryos (ed. R. Bellairs, D.
A. Ede and J. W. Lash), pp. 179-189. New York: Plenum
Press.
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.[CrossRef][Medline]
Morimoto, M., Takahashi, Y., Endo, M. and Saga, Y.
(2005). The Mesp2 transcription factor establishes segmental
borders by suppressing Notch activity. Nature
435,354
-359.[CrossRef][Medline]
Morimoto, M., Kiso, M., Sasaki, N. and Saga, Y.
(2006). Cooperative Mesp activity is required for normal
somitogenesis along the anterior-posterior axis. Dev.
Biol. 300,687
-698.[CrossRef][Medline]
Niwa, Y., Masamizu, Y., Liu, T., Nakayama, R., Deng, C. X. and
Kageyama, R. (2007). The initiation and propagation of Hes7
oscillation are cooperatively regulated by Fgf and notch signaling in the
somite segmentation clock. Dev. Cell
13,298
-304.[CrossRef][Medline]
Oates, A. C., Mueller, C. and Ho, R. K. (2005).
Cooperative function of deltaC and her7 in anterior segment formation.
Dev. Biol. 280,133
-149.[CrossRef][Medline]
Palmeirim, I., Henrique, D., Ish-Horowicz, D. and Pourquie,
O. (1997). Avian hairy gene expression identifies a molecular
clock linked to vertebrate segmentation and somitogenesis.
Cell 91,639
-648.[CrossRef][Medline]
Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J. and
Skarnes, W. C. (2000). An LDL-receptor-related protein
mediates Wnt signalling in mice. Nature
407,535
-538.[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]
Rida, P. C., Le Minh, N. and Jiang, Y. J.
(2004). A Notch feeling of somite segmentation and beyond.
Dev. Biol. 265,2
-22.[CrossRef][Medline]
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C.
(1993). Sonic hedgehog mediates the polarizing activity of the
ZPA. Cell 75,1401
-1416.[CrossRef][Medline]
Saga, Y. and Takeda, H. (2001). The making of
the somite: molecular events in vertebrate segmentation. Nat. Rev.
Genet. 2,835
-845.[CrossRef][Medline]
Saga, Y., Hata, N., Koseki, H. and Taketo, M. M.
(1997). Mesp2: a novel mouse gene expressed in the presegmented
mesoderm and essential for segmentation initiation. Genes
Dev. 11,1827
-1839.
Sawada, A., Shinya, M., Jiang, Y. J., Kawakami, A., Kuroiwa, A.
and Takeda, H. (2001). Fgf/MAPK signalling is a crucial
positional cue in somite boundary formation.
Development 128,4873
-4880.
Schnell, S. and Maini, P. K. (2000). Clock and
induction model for somitogenesis. Dev. Dyn.
217,415
-420.[CrossRef][Medline]
Serth, K., Schuster-Gossler, K., Cordes, R. and Gossler, A.
(2003). Transcriptional oscillation of lunatic fringe is
essential for somitogenesis. Genes Dev.
17,912
-925.
Shifley, E. T. and Cole, S. E. (2007). The
vertebrate segmentation clock and its role in skeletal birth defects.
Birth Defects Res. C Embryo Today
81,121
-133.[CrossRef][Medline]
Sparrow, D. B., Chapman, G., Wouters, M. A., Whittock, N. V.,
Ellard, S., Fatkin, D., Turnpenny, P. D., Kusumi, K., Sillence, D. and
Dunwoodie, S. L. (2006). Mutation of the LUNATIC FRINGE gene
in humans causes spondylocostal dysostosis with a severe vertebral phenotype.
Am. J. Hum. Genet. 78,28
-37.[CrossRef][Medline]
Szeto, D. P. and Kimelman, D. (2006). The
regulation of mesodermal progenitor cell commitment to somitogenesis
subdivides the zebrafish body musculature into distinct domains.
Genes Dev. 20,1923
-1932.
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]
Takahashi, Y., Yasuhiko, Y., Kitajima, S., Kanno, J. and Saga,
Y. (2007). Appropriate suppression of Notch signaling by Mesp
factors is essential for stripe pattern formation leading to segment boundary
formation. Dev. Biol.
304,593
-603.[CrossRef][Medline]
Truett, G. E., Heeger, P., Mynatt, R. L., Truett, A. A., Walker,
J. A. and Warman, M. L. (2000). Preparation of PCR-quality
mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT).
Biotechniques 29,52
, 54.[Medline]
Wahl, M. B., Deng, C., Lewandoski, M. and Pourquié,
O. (2007). FGF signaling acts upstream of the NOTCH and WNT
signaling pathways to control segmentation clock oscillations in mouse
somitogenesis. Development
134,4033
-4041.
Whittock, N. V., Sparrow, D. B., Wouters, M. A., Sillence, D.,
Ellard, S., Dunwoodie, S. L. and Turnpenny, P. D. (2004).
Mutated MESP2 causes spondylocostal dysostosis in humans. Am. J.
Hum. Genet. 74,1249
-1254.[CrossRef][Medline]
Zhang, N. and Gridley, T. (1998). Defects in
somite formation in lunatic fringe-deficient mice.
Nature 394,374
-377.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Wilson, I. Olivera-Martinez, and K. G. Storey Stem cells, signals and vertebrate body axis extension Development, May 15, 2009; 136(10): 1591 - 1604. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
