Aurora B is required for programmed variations of cytokinesis during morphogenesis in the C. elegans embryo

While cytokinesis has been intensely studied, how it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant C. elegans embryo lineage and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we find several unexpected alterations including migration of midbodies to the apical surface during epithelial polarization in different tissues. Aurora B kinase, which is essential for several aspects of cytokinesis, remains localized to the apical membrane after internalization of other midbody components. Inactivation of Aurora B causes cytokinesis failure, which disrupts polarization and tissue formation. Therefore, cytokinesis shows surprising diversity during development and is required during epithelial polarization to establish cellular architecture during morphogenesis.


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Generation of a multicellular organism requires that carefully orchestrated cell 45 division is integrated properly into different developmental processes. Cell division is 46 required not only to generate new cells that organize into tissues, but also to dictate 47 the size, position and timing of daughter cells that are generated. Several aspects of 48 cell division, including spindle orientation and division symmetry are well known 49 instruments of developmental programs (Siller and Doe, 2009). Roles for cytokinesis 50 in regulating developmental events are emerging, but are much less understood (Chen 51 et al., 2013; Herszterg et al., 2014;Li, 2007). Using advanced live imaging, we sought 52 to investigate cytokinesis in the well-defined divisions of the invariant C. elegans 53 embryo lineage, which has been completely described (Sulston et al., 1983). 54 Cytokinesis is the final step of cell division and is normally a constitutive 55 process during the exit from mitosis defined by discrete steps that occur during "C

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Cytokinesis in the first two mitotic divisions: asymmetric midbody inheritance 150 We sought to systematically examine cytokinesis using lattice light sheet and 151 spinning disc confocal microscopy during the stereotypical divisions of the C. elegans 152 embryo, which has been extensively studied primarily in the first cell division due to 153 its size and ease of access. The first mitotic division of the P0 cell generates the larger 154 anterior daughter AB and the posterior daughter P1 (Fig. 1 A). We observed different 155 components that allow us to evaluate specific aspects of the cytokinetic apparatus 156 including the central spindle, the cytokinetic furrow and the midbody. We also chose 157 midbody markers that localize to the flank and ring sub-structures of the midbody 158 (Green et al., 2012). To observe the midbody flank region, we imaged Aurora B 159 kinase (AIR-2), microtubules, and the membrane trafficking regulator RAB-11 ( Fig. 1,   160 3, and Video S1). We also imaged midbody ring markers including the non-muscle 161 myosin NMY-2, which also labels the contractile ring, and the centralspindlin  ). On average, the furrow symmetry parameter is 1.7 in the first division, while 185 the AB furrow is 21.6 and the P1 furrow is 16.1, indicating highly asymmetric 186 furrows in the second divisions ( Fig. 1 V, X). The central spindle is swept from the 187 middle of the AB cell into contact with EMS during furrow ingression ( Fig. 1 E,   188 Video S1). AIR-2 localizes to the central spindle, then the midbody flank and remains 189 associated with the midbody remnant after it is engulfed ( Fig. 1 D- (Leung et al., 1999). The E8 to E16 division occurs around 280 minutes after the first 218 cleavage, after which cells undergo a mesenchyme to epithelial transition involving 219 epithelial polarization and subsequently organize into a tube (Leung et al., 1999). Our 220 observations demonstrate that these cells are performing a highly modified 221 cytokinesis as they undergo polarization, which to our knowledge has not been 222 previously reported (Fig. 2 A). The E8 cells undergo symmetrical furrowing to 223 produce a centrally placed midbody (Fig. 2 B

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In addition to this midbody migration event, we observed a novel behavior of 252 AIR-2 during E8-E16 cytokinesis. In contrast to the midbody ring components, AIR-2 253 remains localized at the apical midline well after the time that ring components are 254 internalized and polarization is complete (Fig. 2 D

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The anterior and posterior pair of E16 cells (Ealaa, Earaa, Eplpp and Eprpp) 286 undergo one last embryonic division to achieve the E20 intestine stage. In the four 287 central E8 cells that do not divide again, the midbody migrates to the midline at E8-288 E16 as described above. However, the midbodies from the other four E8 cells (Eala, 289 Eara, Eplp and Eprp), which undergo another division, migrate toward the midline but  is sick even at permissive temperature, while wild-type N2, zen-4(or153) and spd-315 1(oj5) embryos are 100% viable when kept at 15 °C (Table 1). Embryos shifted to 316 26 °C after 4.5 hours (corresponding to late E4 to early E8 stages) or 6.5 hours 317 (corresponding to E8-E16 stages) at 15 °C showed significantly increased lethality in 318 both air-2(or207) and zen-4(or153), but not spd-1(oj5) (Table 1), which correlates 319 with the amount of cytokinesis failure observed. The few animals that were able to 320 hatch in air-2(or207) and zen-4(or153) mutants had severe morphogenesis defects 321 (data not shown). Mutant embryos shifted after the completion of all the 322 developmental divisions at the comma to 1.5-fold stage were largely rescued for 323 lethality and hatched at a rate similar to permissive temperature (Table 1). Therefore, 324 these results are consistent with the hypothesis that cytokinesis is essential for the 325 final stages of embryonic development during morphogenesis. 326 Next, we tested whether Aurora B kinase was required for the specialized E8-  H). Disrupted ERM-1 staining was also observed in air-2(or207) embryos, which 378 were shifted at E4-E8 for 4.5-5 hours until the comma stage, indicating that these 379 defects are not resolved later in development (Fig. S5 B, D). Furthermore, the 380 intestine was highly mispositioned within the embryo as revealed by color-coded max markers, such as PAR-3, DLG-1 and IFB-2, were similarly disrupted, localizing to the 384 apical surface but in a disorganized way (Fig. S5 E-J). The zen-4(or153) embryos also 385 had highly penetrant branched and discontinuous apical ERM-1 staining that was 386 mispositioned within the embryo at a lower rate than the air-2(or207) mutants ( Fig. 6 387 E, H and Fig. S5 C-D). The spd-1(oj5) embryos displayed a significant, but lower rate 388 and severity of lumen defects (Fig. 6 F-H) despite having no lethality (Table 1)   Apical midbody migration and AIR-2 apical localization in the pharynx 393 We also noticed migration of the midbody and accumulation of midbody both confocal and lattice light sheet imaging (Fig. 8 C and Video S10-11). These 450 midbodies migrate together into a cluster over a 60-minute time window (Fig. 8 D).

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AIR-2::GFP, RAB-11 and tubulin persist in these clusters (Fig. 8 D, Fig. S7 A-F), 452 while ZEN-4::GFP rapidly disappears during the midbody clustering process (Fig. 8 E   453 and Video S10). Endogenous AIR-2 can detected by staining in these lateral apical 454 clusters ( Fig. S1 K-M). In contrast to ZEN-4::GFP, NMY-2::GFP migrates with the 455 midbody to the cluster and persists at the very tip of the dendrites (Fig. 8 F and Video 456 S10). We observe PAR-6 at the tip of the sensilla cluster, indicating that it is the 457 apical surface of these cells, which accumulates -tubulin::GFP similar to the pharynx 458 and gut (Fig. 8 G, Fig. S6 G-L). We also observe -catenin (HMP-1::GFP) at the   clearly labeled by DiI and appeared normal as expected (Fig. 9 A). In the air-2(or207) 493 mutant, we observed numerous defects in the subset of surviving embryos that did not 494 fail to hatch and became L1 larvae ( Fig. 9 B-E). Animals with no observed DiI 495 staining were more common under longer inactivating conditions in the air-2(or207) 496 mutant ( Table 2). All zen-4(or153) fail to hatch when shifted during E4-E8, 497 preventing analysis of DiI staining (Table 2). When shifted from the E8 stage, the few 498 surviving zen-4(or153) larvae show severe DiI staining defects, which was 499 dramatically reduced if embryos were shifted after the final divisions at the comma-500 1.5 fold stage (Fig. 9 F, Table 2). The spd-1(oj5) animals still had weak defects 501 revealed by DiI staining despite having minimal cytokinesis failures, but never 502 showed a complete lack of staining ( Fig. 9 I, Table 2). These data are consistent with   We observe consistent changes to the symmetry of furrow ingression where the 531 first mitosis is relatively symmetric and the second mitosis is highly asymmetric.

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Previously, the furrow asymmetry in the first division was shown to be a consequence      to shift embryos to non-permissive temperature and observe significant cytokinesis 893 defects by the E8-E16 division, indicating significant reduction of AIR-2 function.