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Embryonic Ca2+ signaling
It has been proposed that Ca2+ signaling, in the form of pulses, waves, and steady gradients, may play a crucial role in
key pattern forming events during early
development[1_3]. Webb and
Miller[4] have suggested that in zebrafish, the
complexity of embryonic Ca2+ signaling mirrors that of the developing embryo. Thus, early Zygotic and Cleavage Period
Ca2+ signaling events take the form of relatively simple intracellular waves. Then during the Blastula to early Gastrula Periods,
there is a transition from intracellular to more complex localized intercellular
Ca2+ wave generation. This is followed, as
gastrulation proceeds, by the generation of more extensive (including pan-embryonic) intercellular
Ca2+ waves, which reflect the more wide-ranging morphological events occurring during this period, such as convergent extension and
subsequent axis and germ layer formation. Evidence is beginning to accumulate to suggest that these Gastrula Period intercellular
Ca2+ waves might help to regulate the
coordinated movement of cells during gastrulation in both
fish[5_7] and amphibian[8] embryos. Once the basic embryonic body plan has been established during gastrulation, it has been proposed that
embryonic Ca2+ signaling then returns to more localized, intercellular events that are associated with the generation of
specific structures such as the somites, tail, and various organ
anlagen[4]. In this review, we consider the generation and
possible function of the Ca2+ transients generated during somitogenesis, a fundamental and highly conserved mechanism
used to establish basic embryonic pattern and form.
Somitogenesis
During vertebrate development, the somites are the first segmented structures to form as the paraxial mesoderm
becomes divided into metameric subunits. The somites subsequently give rise to the dermis, the axial skeleton and the skeletal
muscles. Individual pairs of somites, which are formed symmetrically on either side of the notochord, are
generated in an anterior-to-posterior direction within the presomitic
mesoderm[9,10]. Figure 1 is a schematic illustration of a representative
zebrafish embryo to show the anterior-posterior sequence of somite formation in the trunk.
Brennan et al [11] have separated
somitogenesis into four sequential developmental stages: (1)formation and specification of paraxial mesoderm; (2)
patterning of the paraxial mesoderm to establish segmental units; (3) formation of morphological boundaries between segmental
units; and (4) cellular differentiation within somites. These sequential stages will provide excellent reference points when
describing and discussing a variety of
Ca2+ signaling events that have been reported from different embryonic systems at
different times throughout somitogenesis. From recent molecular and genetic studies, it is clear that many aspects of
somitogenesis are conserved across animal
species[12]. It is, however, also apparent that individual species possess unique
features with respect to their somitogenic processes during this crucial developmental period. It is with this in mind, along
with the small number of species so far examined, that we compare and contrast the various reports of
Ca2+ signaling events associated with somitogenesis. When considering the number of reports relating to
Ca2+ signaling during the four stages of somitogenesis outlined above, most relate to the last step in the process (ie, cellular differentiation within somites), then
decline in numbers back towards the earliest of the somitogenic events. In spite of this, we will proceed by considering the
signaling events in a chronological order with respect to somite development.
Ca2+ signaling during the formation and specification of the paraxial mesoderm
The Ca2+ signaling events that occur during this period of development are the least well studied, and as a result,
perhaps the least well understood. Where they have been visualized, however, they have been associated with
morphogenic movements of the gastrulating embryo that are intimately linked to the establishment of the paraxial mesoderm.
Furthermore, the fact that some of these
Ca2+ signals exhibit characteristics similar to those that occur later, and are
coincident with specific somitogenic events, suggests that
Ca2+ transients generated during this early pre-segmentation
period may indeed play a significant role in subsequent segmentation processes.
The most interesting of these Ca2+ transients are those reported in explants of gastrulating
Xenopus embryos[8], where intercellular
Ca2+ waves were visualized propagating through groups of cells undergoing
convergent extension. Waves were only seen in explants that included the dorsal marginal zone, and were never observed from those that encompassed
either the ventral marginal zone or prospective epidermis. The
Ca2+ waves were reported to arise stochastically with respect
to timing and position, and were often accompanied by a wave of contraction within the tissue. Most of the reported waves
arose in the mesoderm, near the dorsal lip of the blastopore. Although convergent extension is more pronounced in the
notochord, it also occurs in the ventrolateral mesoderm that forms the
somites[13]. Using in situ hybridization to the
muscle-specific marker MyoD, Wallingford et
al[8] also showed that at the late gastrula stages, when the stochastic
Ca2+ waves were detected, elongated arrays of
MyoD (ie, prospective somites) flanked the notochord. In thapsigargin-treated embryos,
however, the stochastic Ca2+ waves were suppressed and the lateral extension
of MyoD on either side of the notochord was
significantly restricted. It was proposed that this was due to a failure of convergent extension. In summary, the data
presented by Wallingford et
al[8] suggest that the
Ca2+ transients visualized in Xenopus
explants are not involved in determining cell fate, but that they do play an important role in controlling convergent extension. Thus, via this latter
mechanism, they may play a role in defining the extent of the forming paraxial mesoderm.
Such propagating Ca2+ waves might prove to be a common feature of vertebrate convergent extension, and thus the
formation of the paraxial mesoderm, as they have also been reported from the marginal, converging zone of the epibolizing
blastoderm in developing zebrafish
embryos[6,7].
Although evidence is accumulating to support a role for
Ca2+ signaling in the specification of ventral cell fates prior to
gastrulation[14,15], to date there is no clear evidence to directly link
Ca2+ signaling to the specification of cell fates in the
paraxial mesoderm.
Ca2+ signaling during patterning of the paraxial mesoderm to establish segmental units
Brennan et al[16] reported that highly localized waves of elevated intracellular
Ca2+ propagated through blocks of anterior
presomitic mesoderm (PSM) cells of approximately one somite¡¯s width just prior to somite formation. The precise role of this
Ca2+ increase was not determined, but they proposed that
Ca2+-mediated cell communication via gap junctions might play
a role in determining cellular boundaries during somitogenesis. Thus, unlike the wider-ranging convergent
extension-related intercellular Ca2+ waves described in the previous section, these more localized waves, which occur just before the
somitic boundaries are established, may help to pre-pattern the paraxial mesoderm in order to establish the subsequent
segmental units.
Using the bioluminescent reporter aequorin,
Créton et al[5] described a different type of
Ca2+ wave associated with pre-patterning the paraxial mesoderm. They reported that an "ultraslow"
Ca2+ wave, moving at ~ 0.07 µm/s, propagated along
the trunk in an anterior to posterior direction and they suggested that it might be correlated in some way with the formation
of the somites and neural keel.
It is interesting to note that there are no reports describing any ordered or regular series of
Ca2+ signals generated in the PSM that correlate with any of the well reported patterns of gene
expression[17]. This suggests that the pre-patterning of the
paraxial mesoderm with respect to gene expression, results mainly from
Ca2+ independent mechanisms.
Ca2+ signaling during the formation of morph ological boundaries between segmental units
Over 20 years ago, Chernoff and
Hilfer[18] presented some of the first evidence to suggest that
Ca2+ signaling might play some role in chick somitogenesis by culturing embryonic trunk explants on vitelline membranes in media that was either
Ca2+-free or which contained various
Ca2+ agonists and antagonists. Whereas omitting
Ca2+ in the culture medium resulted in an inhibition of somite formation and led to tissue dissociation, the application of the
Ca2+ ionophore A23187, promoted the rapid, precocious completion of a new somite pair. Furthermore, treatment with the
Ca2+ antagonists, verapamil and papaverine, gave similar results as the
Ca2+-free experiments and reversibly inhibited somitogenesis. These results
suggested that in the chick, somite formation was
Ca2+-dependent, and the source of
Ca2+ was extracellular. Somitogenesis was
also inhibited in embryos treated with the calmodulin-binding antagonists, chlorpromazine and trifluoperazine, and by
treatment with the microfilament and microtubule poisons, cytochalasin D and nocodazole, respectively. Taken together
these results suggested that in the chick,
Ca2+-dependent somitogenesis was mediated by calmodulin and was dependent
in some manner on a contraction event involving microfilaments that helped to define the anterior/posterior morphological
boundaries between segmental units[18]. To date, unfortunately, there has been no attempt to directly visualize
Ca2+ signaling during chick somitogenesis, and thus follow-up these interesting early experiments.
A series of Ca2+ transients have, however, been visualized in the developing myotome
of Xenopus explants[19]. This represents the most detailed report to date with regards to
Ca2+ signaling during the formation of somitic
furrows. Using the fluorescent reporter fluo-3, Ferrari and
Spitzer[19] examined Ca2+ transients in the intact myotome in stage 23/24 embryos,
when 12 to 15 somites have already formed along the anterior-posterior (A-P) axis. For their analysis protocol, they
distinguished four regions along the A-P axis, starting with differentiated anterior somites (AS), followed by maturing
somites (MS), extending to segmenting somites (SS), and culminating in unsegmented paraxial mesoderm (UPM). This
pattern of somite maturation was reflected in the expression of sarcomeric myosin, which was high in the AS and fell to
undetectable levels in the most posterior region of the UPM. The pattern of
Ca2+ transients detected, however, was seen to
be the inverse of this myosin pattern, as myocytes in the AS produced no
Ca2+ transients, while those in the UPM produced
them with the highest frequency. Furthermore, within SS,
Ca2+ signaling activity was seen to be concentrated at the forming
somitic furrows, where cells that were in the closest proximity to the forming somitic furrow were the most active and
signaling coactivity was seen to be transmitted across the nascent somitic furrow. The
Ca2+ transients did not, however,
appear to be associated or produced by cell movements, as transient production was reported to be normal in the
presence of the microfilament disrupting agent cytochalasin D. It was also demonstrated that the transients were generated
by Ca2+ release from intracellular stores via ryanodine receptors (RyR). Blocking this release with ryanodine disrupted the
formation of somitic boundaries. Ferrari and
Spitzer[19] thus concluded that the spatiotemporal pattern of
Ca2+ transients in the myotome indicated a role in the establishment or maturation of the somitic furrow. In addition, the
in vivo Ca2+ transients from Xenopus
myotome explants closely match transients recorded from cultured
Xenopus myocytes with respect to incidence,
duration and frequency. The latter have been shown to be necessary for myocyte differentiation in dissociated cell
culture[20,21].
A variety of Ca2+ transients have also been reported in the trunk of intact zebrafish embryos during the Segmentation
Period. While the study of trunk-generated
Ca2+ signals in zebrafish is still in its infancy, these reports, as well as our
own unpublished results (referred to subsequently as `Leung
et al, unpublished results¡¯) provide hints that
Ca2+ may play a role in some, as yet unconfirmed, aspect of somitogenesis. Using the bioluminescent reporter aequorin, for example, Créton
et al[5] reported localized intercellular
Ca2+ transients along the trunk of segmenting zebrafish embryos and suggested that
they might play a role in mediating the contraction events that result in the formation of somitic furrows. In addition, and also
using aequorin imaging, Webb and
Miller[4] reported that a number of transient localized
Ca2+ signals appeared in the trunk during the segmentation period in zebrafish and they also suggested that the temporal and spatial characteristics of these
signals might perhaps correlate with some aspects of somite formation. In both the above
reports,[4,5] embryos were imaged from a lateral view using aequorin-based imaging (that has poor resolution in the
z-axis[22]), thus neither group was able to
conclude that Ca2+ transients were generated coincidently with the cutting off of each somite pair. More recently, we have
used aequorin-based imaging to re-examine
Ca2+ signaling during the segmentation period in zebrafish from a dorsal view that
allowed us to visualize both sides of the embryonic mid-line (Figure 2,
Leung et al, unpublished results). The most striking
aspect of our new data regarding the
Ca2+ transients generated during the formation of the first eight somite pairs, was the
fact that unlike the physical process of cutting off somite pairs that occurs in a regular, predictable, and reproducible
sequence[16,17], there was clearly no regular, reproducible pattern to the
Ca2+ transients, both within individual embryos and
when comparing one embryo with another. These
Ca2+ transients were thus stochastic in nature and therefore resemble, in
this respect, those reported during convergent extension (ie, during formation of the paraxial mesoderm) in explants from the
dorsal marginal zone of Xenopus
embryos[8]. We assigned these stochastic
Ca2+ transients the general term of "forming
somite signals" (FSS) as they were generated within the segmented paraxial mesoderm once the anterior/posterior somitic
furrow boundaries had formed. Although stochastic in nature, there was a significant tendency for the FSS to occur within
specific regions of the maturing somites, ie, ~75% of FSS were generated at or where the medial or lateral somitic boundaries
were forming. Figure 2 shows two representative examples of embryos (from both dorsal and lateral views) displaying a
number of FSS. We thus suggested that the medial and lateral FSS might provide an additional level of regulation that helps
to define and maintain the medial and lateral somitic boundaries. The incubation of embryos from the 2-somite to the 6-somite
stage with a variety of Ca2+ channel antagonists indicated that these stochastic
Ca2+ transients were generated by
Ca2+ release from intracellular stores via inositol trisphosphate receptors
(IP3R) and not by release via RyR or from extracellular
sources, via Ca2+ channels in the plasma membrane (Leung
et al, unpublished results). The application of the
IP3R antagonist, 2-APB, for example resulted in somites that were lengthened in the medio-lateral dimension. This therefore is unlike the
situation reported for
Xenopus[19] where
Ca2+ release via RyR were responsible for generating the somitic
Ca2+ transients.
As mentioned above, we suggest that there are several significant similarities between the characteristics (and thus
possible developmental function) of the FSS that we described in early zebrafish somitogenesis (Figure 2) and the
Ca2+ signaling reported during convergent extension
in Xenopus embryos[8]. One of these is the common stochastic nature of the
signals reported with respect to morphological events. This might in part be explained by a characteristic that is unique to the
formation of early somites compared to the later ones. Rostral-caudal differences in somitogenesis have been described in
teleosts such as the zebrafish[23] and the rosy barb,
Barbus chonchonius[24], as well as in
chick[25] and
Xenopus[19]. These differences relate to the degree of post-gastrulation convergence that continues during the formation of the early somite
pairs (Figure 1). This is not an issue with regards to later somite formation, as convergence has by then ceased. However,
in early somite formation there is a continued movement of cells towards the embryonic midline that may, via cell-to-cell
interaction, challenge the integrity of the medial boundary between the maturing somite and the notochord in addition to
complicating the process of defining the lateral boundary once the required number of cells has converged into the forming
somite. We suggest that the degree of these convergence-related challenges may vary between individual somites on either
side of the embryonic midline within a single embryo and when each set of somite pair is cut off, with respect to the degree
of convergence still taking place. Thus, if the FSS play some role in establishing and maintaining these medial and lateral
somitic boundaries, their occurrence (and thus visualization) might well reflect this natural variability within individual
embryos (as well as between embryos) and thus explain their stochastic nature.
We also suggest that the cell movements that occur during convergence might also explain why the FSS are not seen until
after the anterior/posterior somitic furrows are established as these represent restrictive boundaries that prevent the
converging cells from spreading out of the forming somite in either an anterior or posterior direction. The onus, therefore, is then
on maintaining the medial boundary followed, at the right time, by establishing and maintaining the lateral boundary. The fact
that the medial and lateral FSS are not seen on a regular basis indicates that they are not the primary signal responsible for
establishing or maintaining these essential boundaries. It does, however, suggest that they may represent a form of
"policing" signal that is generated in response to occasions when the boundaries are challenged; for example when a cell or group
of cells attempts to cross one of the boundaries or tries to leave the forming somite. The latter might explain the signals
visualized from the middle of somites, or from already formed anterior or posterior boundaries. We did not observe any
examples where a FSS was directly associated with the establishment of the anterior or posterior boundary of a somite, ie, a
signal co-incident with a forming somitic furrow. This suggests that in zebrafish, unlike chick or
Xenopus, this key event during somitogenesis appears to proceed largely via
Ca2+-independent processes.
To test our suggestion that the function of the FSS might be to help establish and maintain the medial and lateral
boundaries of somites, we undertook experiments to modulate these signals via either the photo-release of
Ca2+ (via uncaging
NP-EGTA[26]) or photo-activation of a
Ca2+ buffer (via uncaging of
diazo-2[27]). Uncaging Ca2+ in the PSM of presumptive
somites 3, 4 and 5 resulted in somites shortened along their medio-lateral dimension, whereas, photo-activating a
Ca2+ buffer in a similar region, resulted in the elongation of somites along their medio-lateral dimension (Figure 3; Leung
et al, unpublished results). This precocious and ectopic shortening of the somites by uncaging NP-EGTA to release
Ca2+ supports our suggestion that an elevated domain of intracellular
Ca2+ may indeed act as a "policing signal" that helps to establish the
lateral boundary of the somite when other primary signals fail to invoke a response. We suggest that this is achieved via
Ca2+-sensitive elements that effect cell-to-cell contact and thus restrict cell movement. This, therefore, determines the lateral
border and establishes the final somite dimension. The reverse is the case when it comes to activating the
Ca2+ buffer whereby the imposed inability to generate the proposed policing "stop" signal results in somites that extend beyond their
normal medio-lateral boundaries. Once again, we suggest that this could be mediated via
Ca2+-sensitive elements that regulate the contact characteristics between cells. When cells are free to move, they continue to converge into the forming
somites and thus result in a lateral extension. These preliminary experiments therefore suggest a possible developmental
function for the FSS, which is compatible with their stochastic nature. The fact that uncaging either
Ca2+ or Ca2+ buffer had no effect on the establishment of the anterior/posterior somite boundaries supports our previous suggestion that in zebrafish
these might form via Ca2+-independent mechanisms.
In summary, the evidence to date clearly supports the proposition that
Ca2+ signals may play a role during the formation
of morphological boundaries between segmental units. What their precise function is, however, may vary both between
species as well as the particular somitic boundary in question, ie, the anterior-posterior somitic furrow, or the medio-lateral
somitic boundaries. The different signals that have been described in the paper are summarized in schematic form in Figure
4.
Ca2+ signaling during cellular differentiation
within somites
From both in vitro and in vivo studies, evidence is accumulating from
Xenopus[19_21],
mouse[28_30], chick[31] and
zebrafish[16] to indicate that
Ca2+ signaling plays a role in myofibrillogenesis and the development and differentiation of myotubes
(Figure 5). For example, Ferrari et
al[20] reported that differentiating embryonic
Xenopus trunk myocytes produced spontaneous
Ca2+ transients during a restricted developmental window in primary culture. In addition, they showed that suppressing
these Ca2+ transients disrupted myofibrillogenesis and the formation of sarcomeres. Subsequently, it was reported from
cultured Xenopus embryonic myocytes, that these
Ca2+ transients activated myosin light chain kinase (MLCK) in order to
promote myosin thick filament incorporation into developing
sarcomeres[21]. The involvement of MLCK in embryonic
sarcomere development was supported by in vitro
studies on sarcomere organization during cardiac hypertrophy in neonatal
rats[32]. Furthermore, it was recently reported that spontaneous
Ca2+ transients also appear to regulate patterned actin
assemble during myofibrillogenesis in cultured Xenopus
myocytes[33], where blocking the transients disrupted the assembly
of actin thin filaments along with the associated z-disc affiliated proteins titin and capZ. Together, these data suggest that
spontaneous Ca2+ transients may regulate one or more of the earliest steps in sarcomere differentiation.
Following these cultured myocyte experiments, Ferrari and
Spitzer[19] also examined the
Ca2+ dynamics in maturing somites of
Xenopus explants. They showed that the characteristics of the
Ca2+ transients found in the cells of the maturing somites
were remarkably similar to those found in cultured myocytes and were correlated with myocyte maturation. They also
demonstrated that in Xenopus (unlike the chick), these
Ca2+ transients were generated by
Ca2+ release from intracellular stores. Furthermore, they identified the
Ca2+-release channels involved by showing the functional distribution of both
IP3R-activated stores and RyR-activated stores in intact myotome by eliciting
Ca2+ elevations in response to photo-release of
caged IP3 and superfusion of caffeine. These experiments indicated that as in myocyte culture,
Ca2+ transients in vivo depend on
Ca2+ release mainly from RyR stores, and blocking release from these stores interferes with somite
maturation[19].
Brennan et al[16] recently reported from intact zebrafish embryos, that
Ca2+ signaling may also be required for the later
development of slow muscle fiber formation between stages 16 to 22 h post fertilization (ie, at stages where functional
neuromuscular contacts are first established). They also showed that acetylcholine drives initial muscle contraction and
embryonic movement via the release of intracellular
Ca2+ from RyR. When this activity-dependent pathway was inhibited,
either at the level of the acetylcholine receptor or RyR, this did not disrupt slow fiber number, elongation or migration, but did
affect myofibril organization. They proposed, therefore, that
Ca2+ may be acting via the cytoskeleton to regulate myofibril
organization and thus suggested a critical role for nerve-mediated
Ca2+ signals in the formation of physiologically functional
muscle units during development.
Conclusions
A number of different reports demonstrate that starting at the convergent extension events that precede the segmentation
of the paraxial mesoderm, then during the segmentation process itself, and on through to the differentiation of the somites to
form functional skeletal muscle, a range of different
Ca2+ signals are generated from a variety of
Ca2+ stores by a number of different
Ca2+ release mechanisms. Although much has to be done to fully understand the developmental significance and
function of these Ca2+ signals, it is already clear that they play a required role in controlling the generation of somites and their
ultimate developmental fates. In several of the reports to date, the
Ca2+ signals recorded have been to some degree stochastic.
We thus suggest that such signals perhaps represent a new class of developmental
Ca2+ signaling, where Ca2+ is but one of
several developmental regulators, which act in concert with other somitogenesis signaling pathways, such as the PDGF/PI3K
and Wnt/Ca2+ pathways (reviewed by Ulrich and
Heisen-
berg[34]). Both of these pathways have been clearly demonstrated to be connected with
Ca2+ signaling. Moreover, genes in both pathways (for example the
wnt5 gene in the Wnt/Ca2+ pathway and the
PDGFR-a gene in the PDGF/PI3K pathway) are expressed in somites from the earliest stages of
segmentation[35,36]. Thus, the physiological
Ca2+ imaging data reviewed here, support the current genetic and molecular data, and thus provide additional evidence to suggest that
Ca2+ signaling plays a
significant role in somitogenesis.
Acknowledgement
The review was prepared while Andrew L MILLER was the recipient of a Croucher Senior Research Fellowship.
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