Current Biology 20, 2040–2045, November 23, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.10.024 Report A Model for Cleavage Plane Determination in Early Amphibian and Fish Embryos Martin Wu¨ hr,1,2,* Edwin S. Tan,1 Sandra K. Parker,3 To determine when the orientation of the first cleavage plane H. William Detrich III,3 and Timothy J. Mitchison1,2 is established, we repeated Hertwig’s experiment of com- 1Department of Systems Biology, Harvard Medical School, pressing the embryo, which imposes a cleavage plane normal Boston, MA 02115, USA to the long axis of the cell [5]. Shortly after fertilization, 2Physiology Course 2008, MBL, Woods Hole, MA 02543, USA embryos of the African clawed frog Xenopus laevis were 3Department of Biology, Northeastern University, Boston, MA compressed between glass slides. By fixing at different times, 02115, USA we tested when the spindle axis is determined (Figures 1A and 1B). Immunofluorescence staining of a-tubulin and g-tubulin was used to distinguish cell-cycle stages and to Summary measure the angle between sister centrosomes and the imposed long axis of the cell. In prophase, before nuclear Current models for cleavage plane determination propose envelope breakdown, the intercentrosome axis was already that metaphase spindles are positioned and oriented by accurately positioned, differing from the long axis by only interactions of their astral microtubules with the cellular 4.9 6 2.4 (compare to random orientation of 45). This orien- cortex, followed by cleavage in the plane of the metaphase tation did not improve significantly in metaphase (4.2 6 3.7, plate [1, 2]. We show that in early frog and fish embryos, p = 0.5, Student’s t test). Between anaphase and cytokinesis, where cells are unusually large, astral microtubules in meta- alignment improved significantly (1.4 6 1.1, p = 0.001), phase are too short to position and orient the spindle. presumably because the expanding asters, which we will call Rather, the preceding interphase aster centers and orients telophase asters, begin to contact the cortex and thus sense a pair of centrosomes prior to nuclear envelope breakdown, cellular shape. However, the sister-asters are only able to and the spindle assembles between these prepositioned fine-tune the angle of cleavage and not completely reorient it centrosomes. Interphase asters center and orient centro- (Figure S1A and previous work [6]). Cleavage planes were somes with dynein-mediated pulling forces. These forces oriented with an average of 86.1 6 2.8 relative to the artificial act before astral microtubules contact the cortex; thus, long axis, showing that cleavage planes accurately respect the dynein must pull from sites in the cytoplasm, not the cell centrosome orientation imposed before mitosis. To determine cortex as is usually proposed for smaller cells. Aster shape why cleavage planes tend to cut through the sperm entry point is determined by interactions of the expanding periphery in unperturbed embryos [4], we performed immunofluores- with the cell cortex or with an interaction zone that forms cence after fertilization. We observed that sperm aster expan- between sister-asters in telophase. We propose a model to sion is limited by the nearest cortex (i.e., the cortex near where explain cleavage plane geometry in which the length of the sperm entered), resulting in an aster with an oblate ellip- astral microtubules is limited by interaction with these soid shape with its long axis parallel to a tangent to the cortex boundaries, causing length asymmetries. Dynein anchored at the sperm entry point (Figure S1B). In favorable images, we in the cytoplasm then generates length-dependent pulling could visualize paired centrosomes already oriented along this forces, which move and orient centrosomes. axis by w35 min postfertilization. We propose that the centro- somes preserve this orientation at the center, which serves to Results and Discussion orient the first mitotic spindle and in turn to orient the first cleavage plane. The second cleavage plane is orthogonal to Cleavage furrows initiate at a position equidistant between the first. When we visualized centrosomes in telophase of first two radial arrays of microtubules (asters), which grow out mitosis in Xenopus, we found that sister centrosomes are from sister centrosomes at the end of mitosis. The position already oriented orthogonal to the first mitotic spindle before of these asters depends on the prior position of the metaphase astral microtubules have reached the cortex, which is long spindle. In typical animal cells, the mitotic spindle is positioned before nuclear envelope break down for second mitosis in the center of the cell, and oriented parallel to the cell’s long (Figure 1C). In summary, the positions and orientations of axis, by forces acting on its astral microtubules during meta- both the first and second mitotic spindles are determined by phase [1, 2]. In frog eggs, the sperm enters on the side; its the position of centrosome pairs before mitosis onset, which centrosome nucleates a huge sperm aster. This aster captures in turn are determined by the behavior of centrosomes inside the female pronucleus, and moves it, together with the male interphase (or telophase) asters (in early embryos, where there pronucleus and centrosomes, to a position near the cell is no G1 or G2, interphase and telophase are equivalent). center, where the first mitotic spindle assembles. The spindle Internal imaging of amphibian embryos requires fixation and is small compared to cell size, and its astral microtubules are clearing, because yolk with high refractive index is distributed too short to contact the cortex in metaphase [3]. The first throughout the cells. To image microtubules in living embryos cleavage plane tends to cut through the sperm entry point with large cells, we generated a zebrafish line stably express- [4]. The reason for this is unclear (for an overview of microtu- ing the microtubule-binding domain of ensconsin fused to bule organization in early frog embryos see Figure S4A, avail- three GFPs (EMTB-3GFP) [7, 8]. Zebrafish sperm enters the able online). egg near the animal pole, nucleating a sperm aster that spans the whole cell (excluding the lower, yolk-filled part of the egg) (Figure 2A), and captures the female pronucleus. This aster *Correspondence: [email protected] breaks down at the onset of mitosis, and the first mitotic Spatial Organization of Large Cells 2041 A Prophase (t ~ 70min) Metaphase (t ~ 80min) B n=19 n=14 n=20 n=16 8 6 -centrosome -centrosome 4 2 axisandcell’s long axis 0 Angle between centrosome betweenAngle C Anaphase /Telophase ( ~ 90min) Cytokinesis (t ~ 100min) Figure 1. Spindles Are Positioned and Oriented by Asters Prior to Mitosis Onset (A) Frog embryos were artificially elongated by compression and fixed at different time points. Immunofluorescence against a- (yellow) and g-tubulin (red) allows scoring of centrosome orientation and cell-cycle stage. Note centrosomes are already aligned in prophase before nuclear envelope breakdown. (B) Quantification of average centrosome orientation relative to longest cell axis (angle measured between 0 and 90; random orientation would be 45). Centrosomes are already well aligned (4.9 6 2.4 SD) before nuclear envelope breaks down, as soon as they can be visualized with g-tubulin staining. Align- ment does not improve significantly in metaphase (4.2 6 3.7) or anaphase-telophase (4.9 6 3.8). Once the expanding telophase asters touch the cortex, just before cytokinesis, centrosome alignment is fine tuned significantly (1.4 6 1.1). (C) a-tubulin (yellow) immunofluorescence in a frog embryo at anaphase-telophase of first mitosis, before cytokinesis. Duplicated centrosomes are already aligned parallel to the longest axes of the daughter cells. DNA (blue) follows centrosomes. The scale bars represent 500 mm. See also Figure S1. spindle forms. As in the frog, astral microtubules at metaphase pair orients parallel to the interaction zone, aligning with the are too short to contact the cortex, and the spindle forms long axes of the telophase asters (Figure 2D). Interphase nuclei where centrosomes and DNA were deposited by the sperm follow centrosomes (Figures 1C and 2D), presumably by re- aster (Figure 2B and Movie S1). After anaphase onset, telo- cruiting dynein to their surfaces [9]. The spindles of second phase asters expand dramatically from the separating sister mitosis assemble between the separated centrosomes, centrosomes (Figure 2C). At the plane where the sister telo- shortly after cytokinesis (Figures 2D and 2E). Again, their astral phase asters overlap, a zone of reduced microtubule density microtubules at metaphase are too short to reach the cortex. emerges (Figures 2C and 2D). We will call this region, which We conclude that the position (at the cell center) and orienta- we also observed in frog embryos (Figures 1A and 1C), the tion (orthogonal to the first mitotic spindle) of second mitotic aster-aster interaction zone. It seems to form because inter- spindles in zebrafish are determined by the prior position of penetration of microtubules from the two asters is blocked centrosome pairs in prophase, which was in turn determined by unknown mechanisms. in some way by the geometry of the telophase asters they The interaction zone limits the length of microtubules nucleated in the preceding interphase. Because microtubule growing toward the sister aster, creating a length asymmetry organization in fish and frog embryos were similar, we in the left to right direction in Figure 2 (for quantification of combined their technical advantages to probe the mecha- asymmetry see Figure S2). As the telophase asters expand, nisms that locate and orient centrosomes in interphase and and before astral microtubules reach the cortex, the centro- thus determine cleavage plane geometry. somes at their centers start to move apart, toward a point To determine whether centrosomes are moved by pushing midway between the interaction zone and the far cortex or pulling forces, we depolymerized microtubules in selected (Figures 2B–2E and Movies S1 and S2).