Stowers Institute for Medical Research, 1000E 50th street, Kansas City, MO 64110, USA
Author for correspondence (e-mail:
olp{at}stowers-institute.org)
SUMMARY
A characteristic feature of the vertebrate body is its segmentation along the anteroposterior axis, as illustrated by the repetition of vertebrae that form the vertebral column. The vertebrae and their associated muscles derive from metameric structures of mesodermal origin, the somites. The segmentation of the body is established by somitogenesis, during which somites form sequentially in a rhythmic fashion from the presomitic mesoderm. This review highlights recent findings that show how dynamic gradients of morphogens and retinoic acid, coupled to a molecular oscillator, drive the formation of somites and link somitogenesis to the elongation of the anteroposterior axis.
Introduction
The body axis of vertebrates and cephalochordates is subdivided along the
anteroposterior (AP) axis into repeating segments. This segmental or metameric
pattern is established early in embryogenesis by the process of somitogenesis.
Somites are blocks of paraxial mesoderm cells that give rise to the vertebrae
of the axial skeleton and their associated muscles and tendons, which retain a
metameric pattern. They also yield all the skeletal muscles of the body wall
and limbs, as well as of the dermis of the back
(Brent and Tabin, 2002).
During development, somitogenesis is tightly coupled to axis formation via a
system involving dynamic gradients of morphogens, namely fibroblast growth
factors (FGFs), Wnt proteins and retinoic acid (RA).
In the chick embryo, somitogenesis begins soon after ingression of cells
from the epiblast into the mesodermal layer, through the anterior primitive
streak (Garcia-Martinez and Schoenwolf,
1992; Psychoyos and Stern,
1996
). The most anterior paraxial mesoderm cells form the head
mesoderm, which does not display any overt metamerization
(Freund et al., 1996
;
Jouve et al., 2002
;
Kuratani et al., 1999
). The
production of the trunk paraxial mesoderm then follows that of the head
mesoderm without interruption. The first somitic boundary arises a couple of
hours after the beginning of the ingression of the somitic mesoderm, and the
first somite pair forms directly posterior to the otic vesicle region
(Hinsch and Hamilton, 1956
;
Huang et al., 1997
). From this
moment, a new pair of somite boundaries forms sequentially, adding new
segments along the AP axis. A striking feature of somitogenesis is that these
boundaries form at a pace that is species dependent and takes 90 minutes in
chick, 120 minutes in mouse and 30 minutes in zebrafish in optimal temperature
conditions [although some variations in the pace of somitogenesis along the AP
axis have been reported in the mouse embryo
(Tam, 1981
)].
Once formed, somitic cells progressively differentiate to give rise to five
major cell types: the bone, cartilage and tendons of the trunk; skeletal
muscles of the body; and the dermis of the back. This differentiation process
is regulated by extrinsic signals that originate from the tissues surrounding
the somites (for a review, see Marcelle et
al., 2002), and different somitic compartments emerge along the
dorsoventral and mediolateral axes of each segment. The dorsally located
dermomyotome contains the precursors of dermal cells and skeletal muscles,
whereas bone, cartilage and tendon precursors of the back arise from the
ventrally located sclerotome (Brent et
al., 2003
; Brent and Tabin,
2002
). Along the mediolateral axis, the medial part of the somite
will contribute to the muscles of the back and the lateral one will contribute
to the limbs and body wall musculature
(Ordahl and Le Douarin,
1992
)
Each somite is also subdivided into a rostral and caudal compartments
(Stern and Keynes, 1987).
This rostrocaudal compartmentalization has strong implications for vertebrae
formation. The adult vertebrae directly arise from the sclerotome of somites;
however, one vertebra is not produced from one sclerotome, but rather from the
fusion of the caudal half of the sclerotome of one somite with the rostral
half of the following somite in a process called resegmentation
(Bagnall et al., 1988
;
Christ et al., 1998
) (reviewed
by Saga and Takeda, 2001
).
Therefore, the embryonic segments, the somites, and the adult segments, the
vertebrae, are offset by a half-segment, much like the segments and
parasegments in Drosophila
(Lawrence, 1992
). By contrast,
each axial myotome derives from a single somite, which results in the muscles
of the back being attached to two successive vertebrae, allowing the axial
skeleton to bend. This rostrocaudal subdivision is already established in the
anterior presomitic mesoderm (PSM) (Stern
et al., 1991
) and restricts the migration of neural crest cells
and motor axons within the rostral region of the sclerotome, resulting in the
segmentation of the peripheral nervous system
(Bronner-Fraser, 2000
).
Finally, the somitic mesoderm ultimately becomes patterned into cervical,
thoracic, lumbar, sacral and caudal regions. This regionalization is
established early on in the PSM and mostly relies on the activity of Hox genes
(Kieny et al., 1972;
Nowicki and Burke, 2000
)
(reviewed by Krumlauf, 1994
).
Although several links have been established between somitogenesis and the
nested expression domains of Hox genes in the paraxial mesoderm
(Cordes et al., 2004
;
Dubrulle et al., 2001
;
Zakany et al., 2001
), the
coordination of these two patterning processes is poorly understood and will
not be discussed further in this review.
Here, we compare the somitogenesis process between vertebrates and discuss the molecular mechanisms involved in the generation of the metameric pattern from an initial uniform field of cells, the PSM. Somitogenesis is also embedded into the global formation of the AP axis of the developing embryo, and the rate of axis elongation and of somite formation have to be finely balanced. Recent findings suggest that dynamic gradients of RA and FGF/Wnt link somitogenesis to axis elongation.
An overview of somitogenesis
During somitogenesis, at the body level, the paraxial mesoderm consists of
the somitic region anteriorly and of the unsegmented PSM posteriorly. Because
new somites are constantly added during somitogenesis, the ratio of segmented
mesoderm over unsegmented mesoderm increases over time. While somite formation
periodically removes unsegmented material from the PSM anteriorly, new
mesodermal cells are added at the posterior extremity of the unsegmented
tissue by the ongoing gastrulation process taking place in the primitive
streak and later on in the tailbud. However, the net balance between the
removal and addition of unsegmented tissue is not null and the length of the
PSM varies during development. In mouse and chick embryos, PSM formation
starts soon after the beginning of primitive streak regression. Its length
progressively increases and peaks 1 day later, when it contains 12-14
presumptive somites in the chick embryo and around six somites in the mouse
embryo (Packard and Meier,
1983; Tam and Beddington,
1986
). The PSM length then gradually decreases until almost no
unsegmented material remains, which coincides with the end of axis elongation.
However, of these which ends first is unclear (see
Bellairs, 1986
). Somite
formation lasts for 3 days in avian embryos, until the species-specific number
of somites is reached (52 in the chick).
Two important properties of somitogenesis can be defined at this point.
First of all, it is a sequential and directional process: the first formed (or
eldest) somite is located at the anterior tip of the trunk paraxial mesoderm,
and the last produced (youngest) somite is located more posteriorly. Second,
this is a periodic process, where a new boundary is invariably formed after a
given amount of time. Therefore, counting the number of somites is a very
reliable way to stage an embryo. Somitogenesis also has two other striking
properties. The vertebrate body shows bilateral symmetry; this is true for the
paraxial mesoderm, which lies on both sides of the axial structures, the
notochord and neural tube. This is also true for the position of somite
boundaries, which are located at the same AP level for a given pair of
somites. Finally, somite boundary formation is not only periodical but also
synchronous, each somite of a pair being formed simultaneously, indicating
that the development between the left and right side is tightly coordinated.
These latter properties imply that the number of somites on each side of the
embryo is absolutely the same at any given time point. The mechanisms that
control this left/right symmetry are poorly understood. There are, however,
some exceptions among chordates: in Xenopus, for example, even if
somites are symmetrically arranged, their formation can be asynchronous
(Li et al., 2003); in
Amphioxus, somitogenesis is more advanced on the left embryonic side, i.e. it
often contains one more somite than the right
(Minguillon and Garcia-Fernandez,
2002
; Schubert et al.,
2001
).
Each cell contributing to the paraxial mesoderm undergoes a series of
stereotypical events, from its specification in the caudal part of the embryo
to its incorporation into a somite (Fig.
1A). The first step in paraxial mesoderm formation is a change in
the cellular adhesion properties. Cells contained in the primitive streak and
fated to become paraxial mesoderm lose their epithelial characteristics and
become free to migrate laterally to enter the nascent PSM. The forming caudal
PSM thus resembles a loose mesenchymal tissue
(Fig. 1B). In the rostral third
of the PSM, the nuclei of cells occupying the dorsal and ventral aspects of
the tissue begin to align close to the ectoderm and endoderm that,
respectively, overlie and underlie this tissue
(Duband et al., 1987). In the
rostralmost PSM, epithelial characteristics can be easily detected in the
cells that form the future somitic walls. By contrast, cells located in the
middle of the anterior PSM retain a mesenchymal character and will eventually
contribute to the somitocoele mesenchyme that fills the somite cavity
(Fig. 1B). The final step
requires the formation of the somitic cleft, leading to the individualization
of the somite.
|
Genesis of the paraxial mesoderm
Because somitogenesis starts while the formation of the axis is far from being completed, the population of PSM cells has to be continuously renewed. The PSM is constantly supplied caudally with new cells by progenitors located in the primitive streak and tailbud. This section summarizes the molecular events that control the rate of PSM production, the regulation of which is crucial for somitogenesis.
Depending on their position along the embryonic axis, paraxial mesodermal
cells originate from two related embryonic structures. In amniotes, the head
mesoderm and the anterior somites (i.e. those in the occipital, cervical and
thoracic regions) are formed by the ingression of the caudal epiblast through
the anterior primitive streak
(Garcia-Martinez and Schoenwolf,
1992). There is a direct correlation between the timing of the
ingression of a cell and its future location along the AP axis: the later, the
more posterior. After the completion of primitive streak regression around the
16-somite stage in chick, all the tissues contributing to the lumbar, sacral
and caudal regions are produced by a particular embryonic structure, the
tailbud, which forms after the closure of the neuropore at the posterior tip
of the embryo (Catala et al.,
1995
). The tailbud is a small mass of highly packed
undifferentiated cells, which undergo complex stereotyped movements similar to
gastrulation before contributing to the definitive layers. These features
suggest that the tailbud corresponds to a functional remnant of the blastopore
or of the primitive streak (Cambray and
Wilson, 2002
; Catala et al.,
1995
; Gont et al.,
1993
; Kanki and Ho,
1997
; Knezevic et al.,
1998
).
From cell-labeling and lineage-tracing experiments, it has been proposed
that the paraxial mesoderm derives from a population of resident paraxial
mesoderm progenitors (PMP) that is located first in the primitive streak and
then in the tailbud (Gardner and
Beddington, 1988; Nicolas et
al., 1996
; Psychoyos and
Stern, 1996
; Selleck and
Stern, 1991
; Stern et al.,
1992
). There is a direct correlation between the localization of
the PMPs along the AP axis of the primitive streak and the tailbud, and their
final contribution to the mediolateral axis of the mesoderm: PMPs located
anteriorly in the primitive streak or tailbud contribute to the medial aspect
of the mesodermal layer, whereas lateral cells of the paraxial mesoderm derive
from more posterior regions of the streak
(Eloy-Trinquet and Nicolas,
2002
; Freitas et al.,
2001
; Psychoyos and Stern,
1996
; Schoenwolf et al.,
1992
; Selleck and Stern,
1991
; Tam, 1988
).
Thus, the final position of paraxial mesodermal cells in the embryonic space
depends both on the time they are produced, which specifies their AP position,
and their location along the primitive streak/tailbud AP axis at the moment of
their specification, which defines their position along the mediolateral
axis.
Three main events take place in the rostral primitive streak and tailbud,
which lead to the generation of the PSM: proliferation, specification and
emigration. The multiple factors that play a role in each of these steps
mainly belong to four pathways, the Wnt, FGF, RA and bone morphogenetic
protein (BMP) pathways. Representative examples of gene mutations in each of
these pathways are summarized in Table
1. The control of cell proliferation and survival in the growth
zone is crucial to maintain the pool of PMPs during axis elongation.
Inactivation of genes thought to regulate cell proliferation and survival in
the tailbud and PSM, such as those of the Wnt and the RA pathway leads to a
truncation of the axis (Abu-Abed et al.,
2003; Greco et al.,
1996
; Lohnes et al.,
1994
; Marlow et al.,
2004
; Niederreither et al.,
1999
; Yamaguchi et al.,
1999a
) (Table 1). A
second crucial step in paraxial mesoderm production from the pluripotent
tailbud cells is their appropriate specification to the paraxial mesoderm
lineage. Genes belonging to the T-box family of transcription factors, to the
BMP4 and to the Wnt pathway have been shown to play an essential role in this
process (Chapman et al., 1996
;
Galceran et al., 1999
;
Herrmann et al., 1990
;
Streit and Stern, 1999
;
Tonegawa and Takahashi, 1998
;
Yamaguchi et al., 1999b
)
(Table 1). Finally, the control
of the emigration of cells from the primitive streak to form the PSM has been
shown to largely rely on the FGF pathway
(Ciruna and Rossant, 2001
;
Sun et al., 1999
;
Yamaguchi et al., 1994
;
Yang et al., 2002
)
(Table 1). However, all these
pathways interact with each other, directly or indirectly, via positive or
negative feedback loops, which makes it difficult to define a specific role
for a given gene in one of these events.
|
Crucial events take place in the caudal PSM that irreversibly commit PSM cells to their definitive segmental fate. Once this segmental commitment has been achieved, the successive molecular steps that lead to somite formation are activated in a tissue-autonomous fashion in the anterior PSM, ultimately leading to the periodic formation of somites. The spatial and temporal information of segment specification acquired by posterior PSM cells relies on a maturation front and a biological clock that have been molecularly characterized in the past few years.
Generating periodicity within the PSM: the segmentation clock
The striking periodicity of somite distribution and production has led to
the proposal of a series of theoretical models that postulate the existence of
an oscillator or clock that acts in the cells of the PSM
(Cinquin, 2003;
Cooke and Zeeman, 1976
;
Lewis, 2003
;
Meinhardt, 1986
;
Primmett et al., 1989
) (for a
review, see Dale and Pourquie,
2000
). In these models, the oscillator sets the pace of the
segmentation process by generating a periodic signal that is subsequently
translated into the periodic array of somite boundaries. The first evidence
for such a molecular oscillator, termed the segmentation clock, came from the
observation of the periodic expression in PSM cells of chick Hairy1,
a basic helix-loop-helix (bHLH) transcription factor belonging to the
Hairy/enhancer of split family. This gene is expressed as a wave sweeping the
unsegmented mesoderm in a posterior to anterior fashion, once during each
somite formation (Fig. 2B)
(Palmeirim et al., 1997
). A
growing number of genes that exhibit a seemingly dynamic expression pattern in
the PSM, called `cyclic genes', has now been characterized in fish, frog,
birds and mammals, suggesting that the segmentation clock has been conserved
in vertebrates. All identified cyclic genes thus far belong to the Notch and
Wnt signaling pathways. These genes establish interacting feedback loops that
all act downstream of Wnt signaling and generate oscillations in signaling
activities (Aulehla et al.,
2003
; Bessho et al.,
2003
; Dale et al.,
2003
; Hirata et al.,
2004
). The newly identified receptor tyrosine phosphatase
is
also involved in the control of the Notch-driven oscillator
(Aerne and Ish-Horowicz, 2004
).
The role of the segmentation clock in somitogenesis still remains unclear.
However, an important output of the oscillator is the periodic activation of
the Notch signaling pathway in the PSM, which probably plays a crucial role in
the initial definition of the segmental domain, as discussed later. As the
molecular mechanisms of the segmentation clock machinery have been extensively
reviewed elsewhere (Lewis,
2003
; Pourquie,
2003
; Rida et al.,
2004
), we do not discuss them further here.
|
In the chick embryo, rotating a one-somite-length group of cells by
180° in the anterior PSM produces somites with a reversed rostrocaudal
compartmentalization and/or generates segmentation defects (e.g. ectopic
boundaries), suggesting that the position of somitic boundaries is already
determined in this region. By contrast, such rotations in the caudal PSM
result in a normal segmentation pattern, suggesting that these cells are still
naive with respect to their segmentation fate
(Dubrulle et al., 2001). The
interface between these two regions of the PSM has been called the
determination front, which correlates with cellular and molecular changes and
flags the beginning of the segmental determination of the PSM
(Dubrulle et al., 2001
). The
relative position of this front in the PSM is constant, but because of the
anterior-to-posterior progression of somitogenesis, its absolute position
along the AP axis of the embryo is constantly shifted caudally at a velocity
similar to that of somitogenesis.
The position of the determination front has been proposed to be defined by
a threshold activity of FGF signaling. Fgf8 transcripts are
distributed along a caudorostral gradient in the posterior PSM (Figs
2 and
3), which is converted into
graded FGF8 protein distribution (Dubrulle
and Pourquie, 2004). Disrupting the FGF8 gradient by
overexpressing Fgf8 in the chick paraxial mesoderm blocks
somitogenesis and causes cells to retain a mesenchymal character and to
maintain the expression of caudal PSM markers, such as brachyury. Together
with the graded distribution of FGF8 along the AP axis of the PSM, these
results indicate that caudal PSM cells are maintained in an immature state by
high levels of FGF signaling, and that they only activate their segmentation
program when they reach a specific threshold of FGF activity
(Dubrulle et al., 2001
).
|
These data are consistent with a model in which the number of cells
allocated to a given segment is defined by the number of PSM cells
experiencing the passage of the front during one period of the segmentation
clock. Based on this model, somite size can be altered by changing the speed
of the regression of the front or the period of the clock. Although the
involvement of these two parameters, a clock and a sweeping front to specify
segments, was first postulated by Cooke and Zeeman
(Cooke and Zeeman, 1976), the
molecular nature of what generates a threshold most probably falls into a
`clock and gradient' model, as proposed by Meinhardt and Slack
(Meinhardt, 1986
;
Slack, 1991
). However, the
essence of these models is the same. Interestingly, gain- or loss-of-function
gene mutations in mice that affect the segmentation clock [such as those in
lunatic fringe (Lfng) or Notch genes] result in a chaotic spacing of
somite boundaries (Conlon et al.,
1995
; Dale et al.,
2003
; Serth et al.,
2003
). It is tempting to interpret these findings as a
deregulation of the period of the clock, which leads to enlarged somites when
the period is lengthened and to smaller ones when it is shortened.
RA signaling is the second known pathway that regulates the spatial
arrangement of somite boundaries. The first evidence that RA signaling is
involved in the positioning of somite boundaries came from the analysis of
vitamin A-deficient (VAD) quail embryos, which do not synthesize RA. These
embryos display significantly smaller somites than do control embryos, while
the length of their PSM is increased: a phenotype similar to that seen in
response to the grafting of a FGF8-soaked bead into chicken and zebrafish
embryos (Diez del Corral et al.,
2003; Maden et al.,
2000
). Conversely, treating Xenopus embryos with RA leads
to the formation of enlarged somites, a phenotype similar to that seen in
chicken and zebrafish embryos in which FGF signaling has been blocked by
treating them with SU5402, an inhibitor of FGF signaling
(Dubrulle et al., 2001
;
Moreno and Kintner, 2004
).
Although RA cannot be directly detected in the embryo, its distribution can be
deduced by the expression of enzymes that are associated with its metabolism.
Raldh2 (Aldh1a2 Mouse Genome Informatics) an
aldehyde dehydrogenase-like enzyme that converts retinaldehyde into RA, is
expressed at high levels in the newly formed somites of vertebrate embryos and
at a lower level in the anterior PSM (Figs
2 and
3), whereas Cyp26
(Cyp26a1 Mouse Genome Informatics) an enzyme belonging to the
p450 cytochrome family involved in the catabolism of RA, is strongly expressed
in the tailbud region of the embryo
(Blentic et al., 2003
;
Niederreither et al., 2003
;
Sakai et al., 2001
). These
expression patterns are expected to generate an anterior-to-posterior gradient
of RA in the caudal part of the embryo
(Fig. 3).
The RA gradient is thus in an opposite orientation to the FGF gradient, and
it has been shown that they antagonize each other. In VAD quails, the
Fgf8 expression domain is extended anteriorly, and ectopic FGF8
inhibits Raldh2 expression in the somitic region
(Diez del Corral et al.,
2003). In Xenopus, the formation of enlarged somites
after RA treatment is preceded in the PSM by the precocious activation and the
enlargement of Thylacine1 stripes (the frog homolog of
Mesp2/Meso2, see Fig.
2), which mark the earliest segmental domain in the PSM. These
results suggest that the determination front has been shifted posteriorly in
these experiments (Moreno and Kintner,
2004
). They also indicate that RA controls the positioning of the
determination front by antagonizing FGF signaling
(Fig. 3). In frog, this
antagonistic effect is not directly mediated by a downregulation of
Fgf8 expression, but by activating the mitogen-activated protein
(MAP) kinase phosphatase 3 (MKP3), which negatively regulates the MAP kinase
pathway (Moreno and Kintner,
2004
). Mouse Raldh2-null mutants die early during
development and exhibit axis truncation, and, in agreement with the VAD
phenotype, these mutants display smaller somites
(Niederreither et al., 1999
).
These results together suggest that the progression of the determination front
is regulated by two mutually inhibitory, dynamic gradients: a caudorostral
FGF8 gradient that prevents the initiation of the segmentation program; and a
rostrocaudal RA gradient that relieves this inhibition by antagonizing FGF
activity and/or by directly activating genes involved in the segmentation
process.
Finally, the last known pathway involved in boundary positioning is the Wnt
pathway. Grafting clumps of cells that overexpress Wnt3a to the caudal part of
the chick embryo leads to the formation of small somites in the vicinity of
the overexpressing cells (Aulehla et al.,
2003). In mouse, Wnt3a is strongly expressed in the
growth zone of the primitive streak and tailbud, and it was proposed that,
owing to its diffusion properties and to the elongation of the axis, the Wnt3a
protein is distributed in a caudorostral gradient within the nascent PSM. In
support of this idea, Axin2, a negative regulator of the
Wnt/ß-catenin cascade, which is a direct target of Wnt3a, is
expressed in a gradient in the caudal PSM
(Aulehla et al., 2003
). A
striking feature of Axin2 is that its expression is not only graded
in the posterior PSM but it also cycles out of phase with the cycling genes
that belongs to the Notch pathway. It is proposed that the amount of Wnt
signaling directly controls the amplitude of the oscillations of
Axin2, until Wnt signaling drops below a given threshold where the
Axin2 oscillations stop, relieving the inhibitory effect of
Axin2 on the segmentation program. In the vestigial tail
mutants (see Table 1),
Fgf8 expression is downregulated, suggesting that Fgf8 acts
downstream of Wnt3a. It has been proposed in this set of experiments
that the segmentation clock and the morphogen gradients controlling the
determination front are interegulated via Wnt signaling
(Aulehla and Herrmann, 2004
;
Aulehla et al., 2003
).
The fact that the oscillations driven by the segmentation clock are
arrested by the passage of the determination front is consistent with the
observation that the regulatory elements controlling Lfng expression
are different in the anterior and posterior PSM. The promoter region of this
gene has been studied in the mouse and has been found to have a complex
enhancer organization (Cole et al.,
2002; Morales et al.,
2002
). One regulatory block is strictly involved in the cyclic
expression of Lfng in the caudal PSM, while another one is required
for its expression in the anterior PSM.
Accomplishing the segmentation program: from the determination front to somite formation
The accomplishment of the segmentation program the sequence of cellular and molecular events that lead to somite formation in a tissue-autonomous fashion requires complex morphogenetic changes, which occur in the anterior PSM. The key events of this program include cellular reorganization leading to a progressive epithelialization of the tissue, specification of rostral and caudal compartments, and the formation of a somitic cleft between fully patterned blocks of cells (Fig. 1).
Output of the clock and gradients: the Mesp genes?
The immediate consequence of the interaction of the clock and the traveling
wavefront is thought to be the definition of a discrete initial segmental
domain anterior to the determination front level in the anterior PSM, which
will be used as a template for subsequent somite formation
(Fig. 1A). The first genes
expressed in a segmental fashion in the PSM belong to the Mesp family of bHLH
transcription factors (Fig.
2C), which have been characterized in fish, frog, birds and
mammals (Buchberger et al.,
1998; Jen et al.,
1999
; Saga et al.,
1997
; Sawada et al.,
2000
; Sparrow et al.,
1998
; Takahashi et al.,
2000
). In mouse, Mesp2 is activated by periodic Notch
signaling in the rostral PSM in a segment-wide domain, where it controls
downregulation of Delta-like 1 (Dll1), a mouse homolog of the Notch
ligand Delta, in a presenilin-independent fashion
(Takahashi et al., 2000
). The
Xenopus homolog of Mesp2, Thylacine1, is also expressed in
response to periodic Notch signaling in the anterior PSM, and its
transcriptional activation requires direct RA signaling, thus explaining why
it can be activated only after the passage of the determination front
(Jen et al., 1999
;
Moreno and Kintner, 2004
).
These observations have led to the idea that Mesp genes respond to the
segmentation clock and thus participate in translating the periodic signal of
the oscillator into a linear array of segmental domains.
In addition to their role in the initial definition of the segment, the
Mesp genes are also involved in establishing the rostral and caudal
compartment identities of the future somites
(Saga et al., 1997;
Takahashi et al., 2000
). In
the anterior PSM, Mesp genes expression become restricted to half-segment
domains. In mouse, Mesp2 becomes restricted to the future rostral
somitic half and it acts downstream of Notch signaling to specify the rostral
compartments of somites (Saga et al.,
1997
).
Maturation steps within the anterior PSM
Concomitantly to the rostrocaudal compartmentalization of the paraxial
mesoderm, the anterior PSM undergoes a progressive maturation that ultimately
leads to somite formation. Several factors control this maturation, and their
loss of function usually leads to aberrant somite morphogenesis, or to the
failure of somite formation, while segments are specified.
Foxc winged helix transcription factors have been identified as playing a
crucial role in the control of somite prepatterning in the anterior region of
the PSM, where they are strongly expressed. Mice null for both Foxc1
and Foxc2 display no segmentation of the paraxial mesoderm. In these
mutants, some markers of rostrocaudal compartmentalization and of boundary
formation such as Notch1, Mesp1/2, ephrin B2 (Efnb2) are
absent, whereas others, such as Lfng or Dll1, are abnormally
expressed (Kume et al., 2001).
In zebrafish, the knock down of foxc1a leads to a similar phenotype
(Topczewska et al., 2001
).
These results suggest that the Foxc factors may act as permissive signals for
the segmentation program to proceed. These phenotypes are similar to those
seen in the mutant fish fused somites (a tbx24-null mutant),
in which no boundaries form along the entire AP axis of the embryo
(Nikaido et al., 2002
).
fused somites fish ultimately form a segmented axial skeleton,
suggesting that this mutation does not affect the specification of segments
during somitogenesis (van Eeden et al.,
1996
). However, it has been shown that the later segmentation of
the axial skeleton in fused somites is controlled by signals coming
from the notochord (Fleming et al.,
2004
).
Another morphogenetic process that is likely to be initiated by the passage
of the determination front is the epithelialization of the PSM
(Fig. 1). The
mesenchymo-epithelial transition that occurs concomitantly with the segmental
patterning of the anterior PSM is not well understood, but several adhesion
molecules such as integrins, fibronectins and cadherins are progressively
accumulated in the anterior PSM (Duband et
al., 1987). In the mouse, the inactivation of the paraxis gene
(Tcf15), a bHLH transcription factor, results in a lack of
epithelialization of the paraxial mesoderm
(Burgess et al., 1996
).
Tcf15 is expressed rostral to the determination front level in the
anterior PSM and somites (Fig.
2D) (Burgess et al.,
1995
; Sosic et al.,
1997
). Despite the lack of epithelialization in Tcf15
mutant mice, the paraxial mesoderm retains some degree of metameric
organization, even though, at later stages, vertebrae and dorsal root ganglia
are fused (Johnson et al.,
2001
). An outcome of Tcf15 function might be to control
the activity of Rho GTPases such as Rac1 and Cdc42, which have been recently
shown to mediate the mesenchymo-epithelial transition during somite formation
(Nakaya et al., 2004
).
Once PSM cells have been allocated to a given metamer and have acquired
their rostrocaudal identity, the last step in somitogenesis is to create an
acellular somitic boundary. Several signaling pathways have been shown to play
a key role in this process. During somite boundary formation in chick, the
receptor Notch1 is expressed in the forming somite, its ligands
Delta1 and Serrate1 are expressed in the posterior
compartment of the forming somite, whereas Lfng, a
glycosyl-transferase that modulates Notch signaling, is expressed in the
anterior compartment (Sato et al.,
2002). This situation is very reminiscent of the fly wing imaginal
disc, where Notch signaling establishes the boundary between the dorsal and
ventral compartments (Panin et al.,
1997
). Grafting cells from the region lying immediately posterior
to the presumptive boundary of the next-to-be-formed somite (i.e. presumptive
anterior compartment) can induce boundaries in ectopic position (i.e. in the
middle of the somite) (Sato et al.,
2002
). This boundary induction can be mimicked by transplanting
cells that do not normally induce boundaries but that overexpress
Lfng or a constitutively active form of the Notch receptor,
suggesting that modulation of Notch activity via Lfng triggers cleft
formation (Sato et al.,
2002
).
The Eph receptor/ephrin pathway triggers one of the final step of boundary
formation. This pathway controls cell mixing
(Mellitzer et al., 1999) and
is a bi-directional signaling pathway in which both the receptor (Eph) and the
ligand (ephrin) direct downstream signaling events upon activation [called
forward and reverse signaling, respectively
(Murai and Pasquale, 2003
)].
In zebrafish, the ligand and the receptor, in particular ephrinB2 and
epha4 respectively, are expressed on each side of the forming
boundary (Durbin et al.,
1998
). It has been shown in zebrafish that this pathway is
directly responsible for the cellular change that is associated with boundary
formation at the interface between ligand- and receptor-expressing cells: Eph
receptor/ephrin interactions induce cell polarization, the basal localization
of the nuclei, the apical distribution of ß-catenin and columnar shape
acquisition (Barrios et al.,
2003
). Despite the conservation of ephrin expression patterns
between fish and amniotes, no somitic phenotype has been observed in the mouse
Epha4 (Helmbacher et al.,
2000
) or Efnb2 (Wang
et al., 1998
) mutants, possibly owing to the redundant activities
of the ephrins in this species. However, in Notch gene mutants, Epha4
stripes are absent or severely disrupted, suggesting that Notch signaling acts
upstream of Eph signaling during boundary formation
(Barrantes et al., 1999
).
Cell-adhesion proteins also play a crucial role in somite formation, by
regulating the cellular organization of the somitic tissue. Interfering with
the function of cadherins, such as N-cadherin, cadherin 11 or the
protocadherin Papc (Pcdh8 Mouse Genome Informatics)
results in the severe disruption of the epithelialization/somite formation
process (Horikawa et al.,
1999; Kim et al.,
2000
; Kimura et al.,
1995
; Linask et al.,
1998
; Rhee et al.,
2003
).
Finally, the cellular movements and behaviors that lead to cleft formation
have been carefully monitored in chick and zebrafish embryos using time-lapse
confocal microscopy. In chick, the cleft between the forming somite and the
unsegmented mesoderm is not a straight line perpendicular to the axial
structure, but instead appears as a `ball-and-socket': the ball being the
forming somite and the socket the PSM
(Kulesa and Fraser, 2002).
Cells at the posterior edge of the forming somite, after loosing their
adhesiveness with PSM cells, coalesce and move slightly anteriorly. In the
meantime, cells of the dorsal, ventral, medial and lateral aspects of the PSM,
which are in contact with the forming somite, retract and fold in, becoming
the anterior border cells of the next-to-form somite. In zebrafish,
presumptive cells of the anterior and posterior edge of the future border are
first intermixed; they then progressively segregate and eventually face each
other to form the intersomitic border
(Henry et al., 2000
).
Coupling somitogenesis to axis elongation
As discussed earlier, the segmentation of the paraxial mesoderm and the
elongation of the AP axis are highly coordinated during early embryogenesis.
The balance between the rate of somite formation and the production of new
mesodermal cells from the growth zone has to be finely regulated to prevent
the precocious depletion of unsegmented mesodermal material. Recent data
suggest that FGF8 may provide a link between axis elongation and
somitogenesis, via a mechanism relying on mRNA decay
(Dubrulle and Pourquie,
2004).
Fgf8 mRNAs are distributed according to a caudorostral gradient in
the posterior embryo. This mRNA gradient has been shown to be converted into a
graded FGF signaling activity (Dubrulle
and Pourquie, 2004; Sawada et
al., 2001
). The FGF gradient is dynamic, as it recedes in concert
with axis formation. What is the link between the regulation of this gradient
and the formation of the axis? This gradient does not rely upon extrinsic
signals, because the ablation of the tailbud or the in vitro culture of
isolated PSM does not affect the dynamics of the Fgf8 mRNA gradient.
The analysis of regions where Fgf8 is actively transcribed in the
embryo by in situ hybridization in chick and mouse using probes directed
against intronic regions of Fgf8 has revealed that its transcription
is restricted to the tailbud (Dubrulle and
Pourquie, 2004
). Therefore, newly produced mesodermal cells stop
transcribing Fgf8 when they enter the caudal PSM. The pool of
Fgf8 mRNAs within these cells diminishes over time because of mRNA
decay, and, because of the continuous production of PSM cells from the
progenitor area, this patterning strategy leads to the establishment of a
dynamic gradient of Fgf8 mRNAs in the wake of axis elongation. In
agreement with this model, Fgf8 mature transcripts are very stable,
as they can still be robustly detected in the PSM several hours after
treatment with actinomycin D, a broad inhibitor of transcription. Therefore,
the slope of the FGF8 gradient, which eventually regulates the progression of
the determination front, is a direct function of the speed of axis elongation
and of the rate of RNA degradation. The studies of RA previously described
suggest that the extent of the Fgf8 gradient is limited anteriorly by
RA signaling. In contrast to Fgf8, the RA gradient progresses
concomitantly to somitogenesis (and not axis elongation), and it may serve as
a sensor of the rate of somite formation to further regulate FGF signaling.
Whether RA acts directly on the stability of Fgf8 transcripts or
through another mechanism remains to be determined.
It is highly possible that the Wnt3a gradient is also tied to the
elongation of the axis via a similar mechanism. However, in this case, the
axis elongation will not define the slope of the mRNA gradient, but rather the
slope of the protein gradient. In mouse, Wnt3a expression is
restricted to the tailbud, and, although the protein distribution has not been
reported yet, it has been proposed that the continuous production of the
protein from the progenitor cells coupled to the elongation of the axis may
generate a gradient of Wnt3a protein in the caudal part of the embryo
(Aulehla et al., 2003). Wnt3a
might also directly control the transcription rate of Fgf8 in the
progenitors and thus indirectly control the shape of FGF8 gradient, as, in
this model, the extent of the Fgf8 graded expression domain is
directly dependent on the initial amount of Fgf8 mRNAs.
Conclusions
Our current understanding of somitogenesis has greatly improved in the past couple of years. Segmentation appears to rely on two major components: an oscillator, the segmentation clock, which sets the periodicity of somite formation; and a traveling wavefront, which defines the level at which PSM cells respond to the clock, providing a mechanism that spaces the segment boundaries. We propose that the progression of the wavefront is controlled by antagonistic gradients of FGF/Wnt proteins and RA. This mechanism triggers the periodic initiation of the segmentation program in a spatially controlled fashion (Fig. 3). A complex genetic regulatory loop involving Mesp2 and the Notch pathway then subdivides newly specified segments into rostral and caudal compartments. Finally, segments become epithelialized and ultimately separated by a boundary in the rostralmost PSM.
Although great advances have been made in the past decade in understanding
aspects of the somitogenesis process, many things remain to be explained.
First, the molecular machinery that underlies the segmentation clock is far
from understood. It clearly relies on negative feedback loops between
different pathways, but the interactions between these pathways need to be
investigated more deeply. In addition, whether the clock exclusively relies on
the Notch and Wnt pathways or whether it involves a more complex molecular
machinery remains to be established. Moreover, what defines the period of the
clock, and what gives its species specificity is a fundamental question.
Second, the recently discovered involvement of RA in positioning the
determination front and its antagonizing effect on FGF signaling opens up a
new perspective on this process. How this mutual inhibition affects both
gradients is not clearly understood. These findings are especially exciting,
as RA is directly involved in Hox gene regulation
(Conlon, 1995). Finally,
understanding the mechanisms that control the definitive numbers of segments
and the forces that drive the elongation of the axis will provide invaluable
insights into somitogenesis and into the evolution of vertebrate
segmentation.
Note added in proof
Two recent papers published by Hofmann and colleagues and Galceran and
collaborators show that LEF1-mediated Wnt signaling is involved in the
regulation of Delta-like1 in the mouse PSM, supporting the idea of
close interactions between the Wnt and Notch pathways during somitogenesis
(Hofmann et al., 2004;
Galceran et al., 2004
).
ACKNOWLEDGMENTS
The authors thank Marie-Claire Delfini for critical reading of the manuscript. Work in O.P.'s laboratory is supported by the Stowers Institute for Medical Research, by the NIH and by the Muscular Dystrophy Association.
Footnotes
* Present address: Skirball Institute of Biomolecular Medicine, New York
University School of Medicine, 540 First Avenue, New York, NY 10016-6497,
USA
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