Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs

Abstract

The dorsal-ventral axis in frog embryos is specified during the first cell cycle, when the cortex rotates relative to the cytoplasmic core along parallel microtubules associated with the core. Cytoplasmic transfer experiments suggest that dorsal determinants are transported 90 degrees from the vegetal pole to the dorsal equator, even though the cortex rotates only 30 degrees. Here we show that, during rotation, small endogenous organelles are rapidly propelled along the subcortical microtubules toward the future dorsal side and that fluorescent carboxylated beads injected into the vegetal pole are transported at least 60 degrees toward the equator. We also show that deuterium oxide, which broadens the zone of dorsalization even though it reduces the extent of rotation and is known to randomize the microtubules, also randomizes the direction of organelle transport. Moreover, beta-catenin, a component of the Wnt signaling pathway that possesses dorsalizing activity in Xenopus, colocalizes with subcortical microtubules at the dorsal side of the egg at the end of rotation. We propose that cortical rotation functions to align subcortical microtubules, which then mediate the transport of dorsal determinants toward their plus ends on one side of the egg.

Figure 1

Rapid movement of DiOC₆(3)-labeled organelles in the vegetal pole region of an egg during cortical rotation. Optical sections were collected at a frequency of one per second 4–8 μm inside the vegetal surface and then compiled into a time-lapse video. (A and B) The two images shown here were collected 13 sec apart. The red ovals are nile red-stained yolk platelets; between the time of the first image and the second, they have moved toward the upper left at a velocity of ≈10 μm/min. The smaller green circles are DiOC₆(3)-labeled organelles; between the first and second images, most of them have moved toward the upper left (with the yolk platelets), but ≈10% have moved in the opposite direction at ≈30 μm/min. Organelles were continuously tracked through the intervening images. The yellow box (B) outlines the region shown in C and D; the white box in B outlines the region shown in E. (C) Compiled image of a portion of A and B (yellow box, B), at slightly higher magnification, showing change in position of yolk platelets from A (red outlines) to B (purple outlines). (D) Compiled image of a comparable portion of A and B (yellow box, B) at slightly higher magnification showing change in position of DiOC₆(3)-labeled organelles from A (green outlines) to B (blue outlines). The black arrows indicate the routes taken by some of the 10% of small organelles that moved rapidly in the opposite direction of yolk platelet movement. The black boxes in C and D correlate with the white box shown in B. (E) Close-ups taken 1 sec apart in the interval between A and B showing a DiOC₆(3)-labeled organelle (green, white arrowheads) moving from top to bottom and a nile red-labeled yolk platelet (red) moving from bottom to top. By the 12th image, the small organelle has moved more than twice as far as the yolk platelet (Bar = 10 μm.)

Figure 2

Direction of organelle displacements 4–8 μm inside the vegetal surface of immobilized eggs undergoing cortical rotation. Arrowheads indicate the direction of yolk platelet movements that, in control eggs, averaged 11.3 μm. In ²H₂O-treated eggs, yolk platelet displacement ranged between 0.3 and 12.2 μm, with an average displacement of 5.4 μm (n = 12). Arrows show the directions of rapidly moving DiOC₆(3)-labeled organelles. All vectors are brought to a common origin, and the length of the arrows is proportional to the distance traveled by the organelle before it stopped or left the field of view. Only displacements ≥ 3 μm were scored. In control eggs, the rapidly moving organelles are uniformly transported in the opposite direction from the yolk platelets; in ²H₂O-treated eggs, their direction of movement is randomized away from the vegetal pole. Randomized movement also was seen in an additional six ²H₂O-treated eggs (not shown here). (D₂O = ²H₂O.)

Figure 3

Movement of fluorescent beads from the vegetal pole to the equatorial region during cortical rotation. (A) A bolus of beads at the vegetal pole before the onset of cortical rotation. (B) A streak of beads in an egg fixed at the end of cortical rotation, when the first cleavage furrow has begun to form. A linear array of beads is seen extending ≈600 μm from the center of the bolus of beads at the vegetal pole (arrow) toward the equator on the side of the egg opposite the sperm entry point, where the future dorsal side usually forms. Fixed eggs were cut in half along the animal–vegetal axis perpendicular to the cleavage furrow, and each half was compressed between two coverslips (to a final thickness of ≈100 μm) to facilitate visualization of the future dorsal and ventral halves. Optical sections were collected at 0.2-μm intervals, starting at the cell surface and moving into the cytoplasm, to visualize the entire transport zone located 4–8 μm from the cell surface. The final image represents a projection of these sections and shows the entire streak of beads. In some eggs (not shown), beads leaked from the injection needle into the deep cytoplasm between the injection site and the vegetal pole. These streaks were easily distinguished from streaks caused by cortical rotation because they were in the half of the egg containing the sperm entry site, were several hundred microns deeper in the cytoplasm, and were more visible from the bisected cytoplasmic side of the egg than from the plasma membrane. (Bar = 200 μm.)

Figure 4

Colocalization of β-catenin with subcortical microtubules on the future dorsal side of Xenopus eggs at the end of cortical rotation (0.8 of the first cell cycle). Images show labeling with antibodies to β-tubulin and β-catenin (n = 9). (A) Microtubules (green) of the parallel array on the future dorsal side 60–90° from the vegetal pole; (B) linear array of β-catenin (red) in the same region 4–8 μm from the cell surface; (C) colocalization of microtubules and β-catenin (yellow) in this region; (D) microtubules (green) on the future ventral side 60–90° from the vegetal pole; (E) this same (ventral) region shows only small isolated patches of β-catenin; and (F) linear pattern is green rather than yellow (in contrast to C), indicating no colocalization of β-catenin with microtubules on the ventral side. (Bar = 50 μm).

Figure 5

Model of microtubule-mediated rapid transport of dorsalizing components in a free-floating (nonimmobilized) egg. (Left) The highlighted region at the vegetal pole represents a spot [labeled with nile red and DiOC₆(3)] before cortical rotation [Y, yolk platelets; O, small organelles (•, labeled; ○, unlabeled); C, cortex; VP, vegetal pole]. (Right) The location of various components near the end of rotation are shown on the right (MT, microtubules). The core of yolk platelets remains oriented downward, the cortex moves ≈30° along the microtubules, and some organelles in the microtubule transport zone (4–8 μm from the cell surface) move ≈90° along microtubules to the equatorial region on the future dorsal side of the embryo.