Larabell CA et al. (1996), Confocal microscopy analysis of living Xenopus ...

XB-ART-18388

Development 1996 Apr 01;1224:1281-9.

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Confocal microscopy analysis of living Xenopus eggs and the mechanism of cortical rotation.

Larabell CA , Rowning BA , Wells, J , Wu M , Gerhart JC .

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The dorsoventral body axis in amphibian embryos is established by a rotation of the outer cortex relative to the inner cytoplasmic core. This cortical rotation depends on microtubules and is correlated with a parallel array of microtubules just inside the vegetal cortex. Since the parallel array moves with the inner cytoplasm and most of its microtubules are oriented with their plus ends facing the direction of cortical movement, it has been suggested that plus end-directed motor molecules attached to the cortex drive the rotation by moving along microtubules of the parallel array. Using an inverted confocal microscope to examine living eggs, however, we found that rotation movements precede the formation of a detectable parallel array at the vegetal pole, that the parallel array consists of multiple layers of microtubules at depths ranging from 4 to 8 microns inside the plasma membrane and that the velocity of rotation is immobilized eggs increases with depth in this region. These findings suggest that (1) early cytoplasmic movements are due to something other than the fully formed parallel array and (2) the motor molecules responsible for the bulk of the rotation movement are not restricted to a monolayer at the subcortical interface but may be distributed throughout the parallel array, perhaps causing microtubules to slide along other microtubules by a mechanism similar to that seen in cilia and eukaryotic flagella.

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	Fig. 1. Velocity of yolk platelets throughout cortical rotation, in a plane perpendicular to the animal-vegetal axis and 8 mm inside the plasma membrane, at the vegetal pole of a fertilized, immobilized egg. Four phases of cortical rotation can be distinguished: (1) initiation - onset of slow movements between 0.28 and 0.40 NT; (2) acceleration - increase of velocity between 0.40 and 0.50 NT; (3) translocation - continuation of maximal velocity between 0.50 and 0.85 NT, as the cortex undergoes the bulk of its displacement relative to the inner cytoplasmic core; and (4) termination - rapid deceleration after 0.85 NT, sometimes followed by slight reversal of direction.
	Fig. 2. Single optical sections 8 mm inside the plasma membrane at the vegetal pole of a fertilized, immobilized egg, showing the initial slow displacement of Nile Red-labeled yolk platelets between (A) 0.27 NT and (B) 0.45 NT. Yolk platelets have traveled an average of 18 mm in the 14.3 minute time interval between the images; the black arrow indicates the earlier position and the white arrow the later position, of one representative yolk platelet. Bar, 12 mm. 1284 we began our measurements where yolk platelet movements could first be detected (approximately 4-5 mm inside the plasma membrane, as shown in the optical section perpendicular to the coverslip in Fig. 5), then we continued to take measurements at increasing depths until fluorescent platelets could no longer be detected (approximately 14-15 mm inside the plasma membrane). In one egg for which we obtained extensive data, we found that the average velocity of small to medium-sized yolk platelets (£5 mm diameter) in the most peripheral portion of the moving yolk mass (that region about 4 mm from the egg surface) was 4.1±0.5 mm/minute, while those approximately 6 mm from the cell surface moved at an average velocity of 8.1±0.8 mm/minute. (The deviation reported for each of these figures represents the variability among measurements obtained from different yolk platelets in the same egg.) Movement near the interface between the cortex and the cytoplasmic core (at a depth of about 4 mm) was quite turbulent, with stationary organelles being jostled by those moving past them. Some smaller organelles in this interface zone exhibited sporadic movements, with short bursts interrupted by quiescent periods. At greater depths (8-14 mm inside the plasma membrane), medium- to large-sized yolk platelets (>5 mm diameter) were plentiful and tended to move more smoothly than the small yolk platelets at shallower depths, so we used them for all distance measurements at depths greater than 6 mm. In a zone approximately 8 mm deep, yolk platelets moved at a peak velocity (during the translocation phase) averaging 10.7±0.2 mm/minute. Yolk platelets deeper than 8 mm moved with an average velocity which was not significantly different from that seen at 8 mm (Fig. 6). Analyses performed with other eggs, both fertilized and electrically activated, showed a similar increase of velocity with depth. Velocities in other fertilized eggs (n=5) varied from 5.0±1.0 mm/minute at a depth of 4 mm to 10.5±1.0 mm/minute at a depth of 8 mm. (The deviation reported for each of these figures represents the variability among means obtained from different eggs.) In activated eggs (n=3), we found that velocity varied from 5.0±1.0 mm/minute at a depth of 4 mm to 8.5±1.0 mm/minute at a depth of 8 mm, suggesting that variation of velocity with depth may be slightly less pronounced in activated eggs than in fertilized eggs. To verify that the differences that we observed at various depths were not optical artifacts produced by the apparatus, we examined several fixed eggs as described in Materials and Methods. We found that distance measurements made at each depth in these controls were identical to within less than 1%, C. A. Larabell and others Fig. 3. Single optical sections of the region shown in Fig. 2, but at a later time. These images show rapid yolk platelet movement between (A) 0.72 NT and (B) 0.77 NT. Platelets have traveled an average of 23.5 mm in 3.1 minutes. Microtubule bundles of the parallel array can be seen as dark channels among the brightly stained yolk platelets. The microtubules bundles, which in effect are negatively stained, form distinctive patterns (such as the ‘X’ seen here) that can be followed through consecutive images. Specific platelets appear to be consistently associated with specific sites on the bundles, confirming that the inner cytoplasm and the parallel array move together during rotation. Bar, 30 mm. Fig. 4. Single optical section of the same region (8 mm inside the plasma membrane at the vegetal pole) in a fertilized, immobilized egg which was both stained with Nile Red and injected with fluorescein-labeled tubulin. The channels between the Nile Redstained yolk platelets (red) are filled with fluorescein-labeled microtubules (green), confirming that the channels are negatively stained images of microtubule bundles. Optical sections were made at the rhodamine and fluorescein wavelengths simultaneously, then superimposed by computer. Bar, 50 mm. Fig. 5.
	Fig. 3. Single optical sections of the region shown in Fig. 2, but at a later time. These images show rapid yolk platelet movement between (A) 0.72 NT and (B) 0.77 NT. Platelets have traveled an average of 23.5 mm in 3.1 minutes. Microtubule bundles of the parallel array can be seen as dark channels among the brightly stained yolk platelets. The microtubules bundles, which in effect are negatively stained, form distinctive patterns (such as the ‘X’ seen here) that can be followed through consecutive images. Specific platelets appear to be consistently associated with specific sites on the bundles, confirming that the inner cytoplasm and the parallel array move together during rotation. Bar, 30 mm.
	Fig. 4. Single optical section of the same region (8 mm inside the plasma membrane at the vegetal pole) in a fertilized, immobilized egg which was both stained with Nile Red and injected with fluorescein-labeled tubulin. The channels between the Nile Redstained yolk platelets (red) are filled with fluorescein-labeled microtubules (green), confirming that the channels are negatively stained images of microtubule bundles. Optical sections were made at the rhodamine and fluorescein wavelengths simultaneously, then superimposed by computer. Bar, 50 mm.
	Fig. 5. Optical section perpendicular to the plasma membrane (i.e., parallel to the animal-vegetal axis) at the vegetal pole of a fertilized, immobilized egg during cortical rotation. The inner cytoplasm (indicated here by brightly stained yolk platelets) is separated from the plasma membrane by a region 4-5 mm thick which is devoid of yolk platelets. The bright red line at the bottom is the coverslip, which was coated with poly-L-lysine to insure firm adhesion of the egg surface. Bar, 20 mm.
	Fig. 6. Velocity of yolk platelets at five different depths inside the plasma membrane (PM) at the vegetal pole of a fertilized, immobilized egg during cortical rotation. No yolk platelets were detected in the region 0-4 mm inside the PM and confocal images deeper than 14 mm were too faint to be analyzed. In this egg, the velocity of yolk platelets during the translocation period increases with depth from 4 to 8 mm, averaging 4.1±0.5 mm/minute at a depth of 4 mm, 8.1±0.8 mm/minute at 6 mm and 10.7±0.2 mm/minute at 8 mm. (The deviation reported for each of these figures represents the variability among measurements obtained from different yolk platelets.)
	Fig. 7. Polymerization of rhodamine-labeled tubulin near the vegetal pole of a fertilized, immobilized egg, in a region 4-6 mm inside the plasma membrane. Short segments of microtubules are first detected at approximately 0.40 NT (A). Microtubules increase in length and density until 0.54 NT (H). Displacement of yolk platelets in the direction of rotation (movement of black circles from lower left to upper right of each image) can also be seen in these sections (arrows in C and D). Each image represents a single optical section. Normalized times are: A, 0.40; B, 0.42; C, 0.44; D, 0.46; E, 0.48; F, 0.50; G, 0.52; H, 0.54. Bar, 20 mm.
	Fig. 8. Series of single optical sections near the vegetal pole of a fertilized, immobilized egg at 0.65 NT, previously injected with rhodamine-labeled tubulin. The first image (A) shows microtubules approximately 4 mm inside the plasma membrane; each successive image is 1 mm deeper. Due to the spherical shape of the egg, the microtubules visible at the periphery of the deepest sections actually lie at shallower depths. These images show that the parallel array is densest between 4 and 6 mm (AC), and becomes less dense with depth until it is no longer detectable at depths greater than 8 mm (H-I). Bar, 100 mm.
	Fig. 9. Microtubule dynamics during cortical rotation. Optical sections collected very rapidly show lateral movements of microtubule bundles in the parallel array of a fertilized, immobilized egg injected with rhodamine-labeled tubulin. The arrowhead in each frame points to a segment of a microtubule bundle, which undergoes a wave-like lateral displacement of approximately 2 mm in an 18 second time interval. The black dots are pigment granules (»0.25 mm diameter). Bar, 3 mm.