Figure 1.
3D time-lapse series of muscle development in a Ciona intestinalis embryo visualized by electroporation with the sna>Venus plasmid.
(A) gastrula, (B) neural plate, (C) neurula, (D) early tailbud, (E) mid tailbud, and (F) late tailbud stages from a single embryo are shown. The anterior of the embryo is to the left, posterior to the right. A maximum intensity projection of all slices along the z-axis is shown in the xy-plane view for each stage. Cross-sectional slices in the xz and yz-planes are shown above and to the left of the xy-axis view, respectively, and the positions of the slices are represented in the insets. Time stamps are shown in each panel. Scale bars represent 40 µm.
Figure 2.
Schematic illustrations depicting muscle cell development in Ciona and high-resolution imaging of muscle development by dual-tagging muscle cells with spectrally distinct, subcellularly-localized fluorescent proteins.
(A) Schematics of the muscle lineage of the Ciona intestinalis embryo at the one-cell, 8-cell, 32-cell, 110-cell, neurula, early tailbud, mid tailbud and late tailbud stages, with the cell lineages marked by conventional nomenclature. Only one side of the embryo is labeled. Tail muscle precursors are labeled in orange, neural tissue in light blue, trunk mesenchyme in light purple and trunk ventral cells (heart progenitors) in dark purple. Blastomeres that give rise to more than one tissue are stippled with the colors corresponding to their fates. (B–I) Time series of embryos co-electroporated with sna>GPI-GFP and sna>H2B-RFP. (B,F) neurula, (C,G) early tailbud, (D,H) mid tailbud, and (E,I) late tailbud stages are shown. (B–E) A single slice in the z-axis is shown in the xy-plane of view. Cross-sectional slices in the xz and yz-planes are shown above and to the left of the xy-axis view, respectively, and the positions of the slices are represented in the insets. (F–I) A maximum intensity projection of all slices along the z-axis is shown in the xy-plane of view. Scale bars, 40 µm.
Figure 3.
Tracking the position of muscle nuclei during tail extension in wild type and perturbed embryos.
In each case, the same embryo is shown at the early tailbud (left) and late tailbud (right) stages. Nuclei followed for tracking are highlighted with false colors and numbered. The anterior tip of the embryos (origin) and the notch at the intersection of the trunk and the tail, used to correct for movement in the frame, are highlighted in yellow and pink, respectively. Time stamps are shown in each panel. (A) A wild type embryo electroporated with sna>H2B-RFP, and (D) a perturbed embryo co-electroporated with sna>Bix and Tbx6b>H2B-GFP. (B, E) Paths of movement of selected muscle cell nuclei (mn), relative to the position of the anterior tip, in wild type (B) and sna>Bix (E) embryos. (C, F) Change in internuclear distance between nearest neighbor muscle cells during tail extension in unperturbed (C) and sna>Bix (F) embryos. Tail lengths for the sna>Bix (F) embryo were measured in parallel from a stage-matched wild type embryo. Because sna>Bix affects tail extension, staging of sna>Bix embryos was based upon measurements of wild type embryos cultured in parallel from the same batch. Wild type embryos were monitored on a wide-field inverted microscope located directly adjacent to the laser scanning confocal microscope, to ensure that the two samples were maintained in similar environmental conditions. Scale bars, 40 µm.
Figure 4.
Changes in muscle cell geometry during tail extension.
(A) Muscle cell boundaries highlighted by GPI-GFP in an embryo co-electroporated with sna>GPI-GFP and sna>H2B-RFP, shown at early tailbud and late tailbud stages. (B) Average muscle cell shape (ratio of length to height; n = 5) from the sna>GPI-GFP + sna>H2B-RFP embryo plotted against tail length, with standard deviation shown for each data point. (C) Embryo co-electroporated with sna>Bix to perturb muscle development and Tbx6b>GPI-GFP to mark muscle membranes. (D) Graph of average muscle cell shape (ratio of length to height; n = 5) from the sna>Bix + Tbx6b>GPI-GFP embryo plotted against tail length of a wild type embryo monitored in parallel, with standard deviation shown for each data point. (E) Schematic of muscle cell shape changes in wild type and sna>Bix embryos during tail extension. Time stamps are shown in each image panel. Scale bars, 40 µm.
Figure 5.
Muscle cell volume during tail extension.
(A) Cross-sectional slices of GPI-GFP labeled muscle cells at the early tailbud stage. The underlying notochord cells are highlighted by coelectroporation of Bra>RFP [11]. (B) Cross-sectional slices of the same muscle and notochord cells at the late tailbud stage. (C) Graph of cell volumes for three muscle cells during tail extension. (D) Graph of changes in anteroposterior length, dorsoventral height and mediolateral width during tail extension in “muscle cell 1” from the previous panel. Scale bars, 10 µm.
Figure 6.
Differential protrusive activity at the plasma membrane of muscle cells during tail extension.
(A) Neurula, (B) early tailbud, (C) mid tailbud and (D) late tailbud stage embryos electroporated with sna>GPI-GFP and imaged by high-speed spinning disk confocal microscopy. Brightfield images of stage matched embryos are shown in the bottom left corner of each panel. High magnification views of the muscle cells membranes outlined by dashed lines are shown in panels A and B. (E–H) Actin-based cell surface projections imaged by electroporation of Tbx6b>PH-YFP. (I–L) Polar plots of muscle cell protrusion length and orientation in neurula, early tailbud, mid tailbud, and late tailbud embryos. Protrusion length and angle are shown as the distance from the center of the plot and the angle from the x-axis respectively. (M–P) Cell surface projections in perturbed embryos. Embryos were co-electroporated with sna>Bix and Tbx6b>PH-YFP. (Q–T) Polar plots of muscle cell protrusion length and orientation in perturbed embryos co-electroporated with sna>Bix and Tbx6b>PH-YFP. Distance in µm is shown along the x-axis. Scale bars, 20 µm.
Figure 7.
Impairment of muscle cell activity by cytochalasin D.
(A) Brightfield image of a wild type neurula stage embryo, with dashed lines outlining the region shown in high magnification in panels B–D. (B–D) High-speed confocal images of a wild type embryo electroporated with sna>GPI-GFP. Yellow arrowheads highlight dynamic projections, which had a duration of less than two minutes. (E) Brightfield image of a neurula stage embryo incubated in 500 nM cytochalasin D, with dashed lines outlining the region shown in high magnification in panels F–H. (F–H) High speed confocal images of an embryo electroporated with sna>GPI-GFP and treated with cytochalasin D. Blue arrowheads highlight projections, which were static throughout the 5 minute imaging period. Scale bars, 10 µm.
Figure 8.
Three-phase model of cell behavior operating during tail extension in Ciona.
High magnification photomicrographs of muscle cells expressing GPI GFP are shown in the left column. Schematics of wild type muscle cells are shown in the middle column, with the borders and projections of a single cell outlined in red. Schematics of muscle cells expressing Bix1 are shown in the right column. Muscle cells undergo their last division at the neurula stage. As they adopt their stereotypical relative positions they exhibit highly protrusive activity at the lateral plasma membranes. By the early tailbud stage, as cells have initiated elongation, protrusive activity has declined but has become polarized, with the majority of plasma membrane protrusions extending anteriorward. By the mid tailbud stage, elongation is underway and protrusive activity has ceased, with cells appearing almost quiescent. In sna>Bix embryos very few projections are observed in muscle cells and muscle elongation is impeded.