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	Figure 1. Nocodazole-induced spindle depolymerization defines two classes of embryos with qualitatively different mitotic responses. (a) Phylogenetic tree indicating phyla and species. Species analyzed in this study are in black, species for which information was obtained from the literature in grey. (b) Schematic representation of assay used to test mitotic progression. Two-cell stage embryos of selected species were treated either with DMSO or nocodazole and then fixed every 10–15 min for pH3 immunostaining. (c) Percentage of embryos accumulating pH3 in the presence of DMSO (blue) or nocodazole (red) over time (minutes indicated in x axis). Representative of 4 independent experiments, n = 50–200 embryos for each time point. Drugs were added when at least 90% of embryos were at 2-cell stage (t0). For M. galloprovincialis, control could not be quantified past 8 cells as divisions become asynchronous within each embryo. The duration of normal cell-cycle timing for each species is indicated under the corresponding graph.
	Figure 2. Microtubule depolymerization causes an Mps1-mediated mitotic block in cleavage stage embryos of P. lividus, C. hemisphaerica, and M. galloprovincialis. (a) Schematic representation of the effect of reversine on cell cycle progression during spindle assembly checkpoint (SAC) activation (+ nocodazole). (b) Quantification of duration of mitosis in P. lividus embryos treated with DMSO, nocodazole, or nocodazole and reversine. Mitosis was measured as time from nuclear envelope breakdown (NEB) to nuclear envelope reformation (NER). Boxes represent 25th–75th percentiles and the median is shown; whiskers mark 5th and 95th percentiles. Each dot represents one cell. Asterisks indicate statistical significance as determined by Student’s t-test, p < 0.001. (c) Quantification of embryos accumulating pH3 in the presence of DMSO (blue), nocodazole (red) or nocodazole/reversine (green) over time equivalent of two cell cycles. (d) Labeling of newly replicated DNA by 5-ethynyl-2′-deoxyuridine (EdU) incorporation in embryos treated with DMSO (left), nocodazole (middle), or nocodazole/reversine (right). (e) Quantification of duration of mitosis in C. hemisphaerica embryos treated with DMSO, nocodazole, or nocodazole/reversine. Box plot parameters as in (b). (f) Representative DIC images of embryos treated with nocodazole (left) or nocodazole/reversine (right). Arrows point at nuclei visible in DIC optics. White squares indicate nuclei-containing regions at time of NER, enlarged in insets. (g) Quantification of pH3 positive C. hemisphaerica embryos in the presence of DMSO (blue), nocodazole (red), or nocodazole/reversine (green). Representative of 4 independent experiments, n = 20–30 for each time point. (h) Quantification of Nup153-labeled and (i) pH3-labeled M. galloprovincialis embryos after treatment with DMSO (blue), nocodazole (red), or nocodazole/reversine (green). (j) Representative images of embryos stained for Nup153 (top), DNA (Hoechst, bottom), and (k) pH3. PB = polar body. Scale bar = 30 µm.
	Figure 3. P. mammillata 2-cell embryos do not arrest in mitosis in the absence of spindle microtubules. (a) Selected frames from a time-lapse movie of a P. mammillata embryo expressing the DNA reporter, H2B-Venus, treated with 10 µM nocodazole after first cleavage. Numbers indicate minutes after treatment. Arrows indicate nuclei visible in bright field optics. See movie 1. (b) Duration of mitosis (M, NEB to NER) and interphase (I, NER to NEB) in P. mammillata embryos treated with DMSO or nocodazole. Kin/V indicates kinetochore to cell volume ratio at each cell cycle starting from fertilization (1st is 1-cell mitosis; 2nd is 2-cell mitosis). Box plot parameters as in Figure 2b. In nocodazole-treated embryos the amount of DNA increases due to subsequent rounds of DNA replication without intervening cytokinesis. Duration of mitosis in control DMSO-treated embryos is constant in all four analyzed cell cycles. (c) DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride)-stained chromosome spreads from DMSO and nocodazole-treated (180 min) P. mammillata embryos. After 180 min nocodazole treatment, embryos have undergone 4 more cell cycles and have 4 times more DNA than at time 0. In the absence of microtubules, the nuclei which form around chromosome clusters (karyomeres) do not fuse and are dispersed over time by cytoplasmic flow (in (a,c)). Scale bar = 30 µm.
	Figure 4. Nocodazole treatment does not delay mitotic progression during cleavage in chordate embryos. (a) Quantification of Nup153-negative C. intestinalis embryos (without nuclei = mitosis) in the presence of DMSO (blue) or nocodazole (red) over the time of 3 divisions (2–16 cells). Representative of 3 independent experiments. n = 20–30 embryos for each time point. (b) Representative C. intestinalis Hoechst-stained nuclei and (c) Nup-153-stained embryos. (d) Percentage of B. lanceolatum embryos without nuclei, as determined by Nup-153 staining, in the presence of DMSO (blue) or nocodazole (red) over the time of two divisions (2–8 cells). Representative of 3 independent experiments. n = 50–100 embryos per time point. (e) Representative B. lanceolatum Hoechst-stained nuclei and (f) Nup-153-stained embryos. Numbers indicate minutes after treatment. (g) Measurement of long axis of embryo (width, blue) and cell–cell contact region (midline, orange) during 2–3 cell cycles in a representative embryo of B. lanceolatum (Bl), P. mammillata (Pm, see movie 1), C. intestinalis (Ci), and P. lividus (Pl) in the presence of nocodazole (+ noco; movie 2) or for Pl nocodazole/reversine (Pl + noco + rev; movie 3). Measurements are reported as percentage of maximum length throughout the recording. Crosses (×) correspond to NEB, and circles (•) to NER. Scale bar = 30 µm, unless otherwise indicated.
	Figure 5. SAC response across species does not correlate with cell volume. (a–c) Ratio of average mitotic duration in nocodazole- and DMSO-treated embryos plotted against (a) chromosome number, (b) cell volume at 2-cell stage or (c) kinetochore/cell volume ratio at 2-cell stage. Bl = B. lanceolatum, Ci = C. intestinalis, Ce = C. elegans, Pl = P. lividus, Pm = P. mammillata, Pm5 = ratio after 4 cycles in nocodazole from Figure 3b, Ch = C. hemisphaerica and Xl = X. laevis. For Bl and Ci, mitotic duration was estimated from the time course graphs in Figure 4a,d, as width of the curve at 50% level. Ce and Xl data were obtained from the literature. For Ce, as the first division is asymmetric, both cells are presented (AB and P1). r indicates the coefficient of correlation for each set of variables.
	Figure 6. Mad1 does not localize to unattached kinetochores in 2-cell P. mammillata embryos. (a) Mad1 antibody recognizes a single 80 KDa protein in P. mammillata egg extract which is specifically lost (top panel) in competition assays when incubated with purified His-Mad1, but not with the equivalent amount of BSA (15 µg), whereas tubulin levels (bottom panel) are unaffected. (b) Mad1 level is constant in both untreated and nocodazole-treated eggs and 2-cell embryos. (c) Mad2 antibody recognizes a single 23 KDa protein in P. mammillata eggs, 2-cell and 8-cell embryos, and a larger band of the correct size (27 KDa) when tagged Mad2 protein is overproduced by microinjection (left lane: 5 eggs injected with mRNA encoding Mad2 fused to 36 additional amino acids). (d) Representative images of nocodazole-treated eggs (top) and 2-cell embryos (middle and bottom panels) stained for Hoechst (blue), Mad1 (green), and pH3 (red). White squares in (d) indicate chromatin-containing regions enlarged (5×) on the right images. Scale bar = 30 µm.
	Figure 7. Mad2 and Mps1 are not recruited to unattached kinetochores in 2-cell P. mammillata embryos. (a) Representative images of live eggs (top, middle rows) and 2-cell embryos (bottom row) expressing histone H2B-Venus and Mad2-Tomato. (b) Ratio of Mad2-Tomato fluorescence intensity in chromosome region (A) and cytoplasm (B) in nocodazole-treated eggs and 2-cell embryos (c) Representative images of live eggs (top) and 2-cell embryos (bottom) expressing histone H2-Venus and Mps1-Tomato. (d) Ratio of Mps1-Tomato fluorescence intensity in chromosome region (A) and cytoplasm (B) in nocodazole-treated eggs and 2-cell embryos. White squares in (a,c) indicate chromatin-containing regions enlarged (5×) on the right images. Scale bar = 30 µm.

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