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Embryonic Xenopus myocytes generate spontaneous calcium (Ca(2+)) transients during differentiation in culture. Suppression of these transients disrupts myofibril organization and the formation of sarcomeres through an identified signal transduction cascade. Since transients often occur during myocyte polarization and migration in culture, we hypothesized they might play additional roles in vivo during tissue formation. We have tested this hypothesis by examining Ca(2+) dynamics in the intact Xenopus paraxial mesoderm as it differentiates into the mature myotome. We find that Ca(2+) transients occur in cells of the developing myotome with characteristics remarkably similar to those in cultured myocytes. Transients produced within the myotome are correlated with somitogenesis as well as myocyte maturation. Since transients arise from intracellular stores in cultured myocytes, we examined the functional distribution of both IP(3) and ryanodine receptors in the intact myotome by eliciting Ca(2+) elevations in response to photorelease of caged IP(3) and superfusion of caffeine, respectively. As in culture, transients in vivo depend on Ca(2+) release from ryanodine receptor (RyR) stores, and blocking RyR during development interferes with somite maturation.
FIG. 1. Developmental progression of somite formation. (A) Somite morphology assessed in a stage 23 fixed embryo by whole-mount
sarcomeric myosin immunoreactivity. Boxed areas correspond to the four regions of the myotome imaged for Ca21 activity shown in Fig.
2: AS, anterior somites; MS, maturing (middle) somites; SS, segmenting somites; and UPM, unsegmented paraxial mesoderm. Anterior is
to the left in this and subsequent images; myosin expression is gradually reduced from anterior to posterior. Dorsolateral ectoderm and
dermatome were removed to reveal the underlying endoderm and myotome. Image is a z-series maximum projection of 50-mm confocal
sections; bar, 100 mm. (B) Correlation of embryonic age (h) and stage (Nieuwkoop and Faber, 1967) with number of somites. Somite data
were compiled from Hamilton (1969) and Muntz (1975). The linear correlation coefficient for somite number and age is 0.995.
FIG. 2. Spontaneous Ca21 transient activity in the developing myotome. Pseudocolor single frames from time-lapse sequences of fluo-3
fluorescence in somitic cells in the dorsolateral myotome of three different embryos at stage 23/24. Images were captured at 10 or 15 s
intervals. All active cells from the sequence are outlined in white, and somitic borders are indicated by dashed lines. Numbers indicate
transients produced within the 30-min imaging period. (A) Region of maturing somites (MS). Image shows one active cell (yellowâgold) of
12 active cells in this field. In this phase of somitogenesis, somite boundaries are clearly demarcated, but myocytes have not yet fully
elongated to span the somite width. Ca21 transients were not observed in more mature somites in which myocytes are fully elongated (AS
region). (A9) Representative trace of activity over 30 min in cell indicated. (B) Region of somite segmentation (SS). Image shows a group of
coactive myotomal cells that span a forming somitic furrow. A QuickTime movie from the boxed region can be viewed at wwwbiology.
ucsd.edu/;ferrari/Home/MFMovies.html. In this phase of somitogenesis, furrows form to carve out somites approximately 10 cells
long. Ca21 transient activity appears highest at these forming furrows. (B9) Representative traces from this region in the 3 cells indicated.
Some cells appear phase-locked despite their apparent physical separation. (C) Unsegmented paraxial mesoderm (UPM). This area shows
the highest Ca21 transient activity, and transient durations, amplitudes, and frequencies are similar to transients observed at early times
in culture. Note the coactivity between groups of cells. (C9) Representative traces from the two cells indicated. Transients have longer
durations, higher frequency of activity, and greater variability in kinetics compared with those in more mature myocytes. (AâC) Scale bar,
100 mm. (A9âC9) x-axes are 200 s and y-axes are 5 fluorescence units (F; raw pixel intensity). Fluo-3 is nonratiometric and comparisons of
amplitude between cells are not possible.
FIG. 3. Correlation of transient activity with somite furrow formation. (A) Ca21 elevations occur simultaneously in cells on both sides of
furrows (indicated by dashed lines) in the SS region of another stage 23/24 embryo. A QuickTime movie from the boxed region is available
at www-biology.ucsd.edu/;ferrari/Home/MFMovies.html. Bar, 100 mm. (B) There is no correlation between transient frequency and
distance from the somitic furrow in a scatterplot of individual cells from MS and SS regions (same data set used in BâD). (C) Cells at the
most ventral aspect of the exposed myotome generate the highest transient frequencies. (D) The incidence of activity declines continuously
with distance from the somitic furrow, with little activity present in the middle of somites (somites average 100 mm in AâP length).
Histogram displays cumulative counts of active cells in 5-mm bins.
FIG. 4. Physiological measure of developmental expression of IP3 receptors in cultured myocytes. (A) Responses of cells coloaded with
membrane-permeant forms of fluo-3 and caged IP3 at 6 h in culture. (Left) Phase-contrast image of cells; note polarizing myocytes
(arrowheads) and numerous undifferentiated round cells. (Middle) Resting Ca21 levels measured by fluo-3 fluorescence prior to photorelease
of IP3. (Right) Robust Ca21 increases occur in round cells after 2 s UV illumination to photorelease IP3, but myocyte responses (arrows) are
smaller. (B) Digitized traces of round cell and myocyte responses (from cells marked with asterisks in the middle panel of A) to sequential
IP3 challenges at 6 h in culture. Round cells produce large Ca21 transients with a biphasic decay in response to IP3 production. Myocytes
generate smaller transients after photorelease of IP3 and have a single exponential decay. x-axis, s; y-axis, F. (C). Developmental
responsiveness to photorelease of IP3 in myocytes vs round cells in culture. IP3 stores are maintained in round cells, but disappear by 12 h
in myocytes. This time course corresponds roughly to the period of spontaneous Ca21 transient production in cultured myocytes (grey bar).
FIG. 5. Developmental expression of functional IP3 receptors in the intact myotome. (Left) Fluo-3 fluorescence prior to IP3 uncaging.
(Middle) Ca21 elevations induced after a 5-s UV illumination to uncage IP3. (Right) Activation of IP3 receptors is illustrated by the relative
difference in fluorescence pre- and post-IP3 photolysis presented in inverted format. (AâC) IP3 response in the SS and UPM regions of a stage
26 embryo. Ca21 elevations are relatively uniform in both the AâP and DâV axes in response to IP3 release. The distribution of functional
receptors in these regions coincides with the spontaneous production of Ca21 transients. (DâF) Responses in the more anterior MS and SS
regions of another stage 26 embryo. (GâI) Functional IP3 receptors are still present in the AS region of a third stage 26 embryo. (JâL) IP3
sensitivity is absent in more mature somites, as seen in the AS region of a stage 33 embryo, although IP3 responses occur in nonmuscle cells
along somite borders.
FIG. 6. Presence and functional block of RyR-activated stores in the developing myotome. (A, B) Fluo-3 fluorescence in the SS and UPM
regions of a stage 23 embryo before and after perfusion with 40mMcaffeine. Bar, 100 mm. (C) The robust response throughout the myotome
is shown by the absolute difference of pre- and postcaffeine images shown in A and B, in inverted format. (D) Ca21 transient activity in
individual cells after application of 100 mM ryanodine. Cells usually produce only a single transient, followed occasionally by a second of
lesser amplitude, consistent with use-dependent block. (E) Transient production before and after application of 100 mM ryanodine.
Scatterplot of cumulative histogram data (100-s bins) over 30 min for control (squares) and ryanodine-treated (diamonds) embryos.
Ryanodine was applied immediately prior to imaging runs. The exponential decay of transient production following application of
ryanodine (dashed line) is consistent with initial activation and subsequent block of RyR (Meissner, 1986, 1994). In contrast, transient
production is steady in controls (solid line).
FIG. 7. Chronic ryanodine treatment disrupts somite maturation in vivo. (A) Skeletal muscle morphology in normal stage 41 embryo
assessed by sarcomeric myosin whole-mount immunocytochemistry. Note the regularity of somite arrangement and myosin expression
in the extraocular muscles and hypaxial musculature. Image is a z-series maximum projection of 50-mm confocal sections; bar, 500 mm. (B)
Chronic ryanodine (100 mM) from stages 23â41 (48 h) disrupts embryonic development. Prominent features are hydrocephalus and severe
scoliokyphosis of the trunk. Myosin expression levels appear normal in extraocular muscles and throughout the myotome, but somites and
myocyte morphology are disrupted. However, segmentation of the distal somites is apparently initiated normally (arrowheads in expanded
image at right).
