Programmed Variations of Cytokinesis Contribute to 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 lineage of the C. elegans embryo and find that several parameters are reproducibly altered in different stages. During early divisions, cells undergo consistent patterns of furrow ingression asymmetry and midbody inheritance, suggesting specific regulation of these events. During morphogenesis, in the intestine, pharynx, and amphid sensilla, we find several alterations including migration of midbodies to the apical surface during cellular polarization. In each tissue, Aurora B kinase localizes to the apical membrane after internalization of other midbody components. Perturbations of cytokinesis disrupt lumen formation and dendrite formation. Therefore, cytokinesis shows surprising diversity during development, and may regulate the final interphase architecture of a terminally dividing cell during morphogenesis.


Introduction
Generation of a multicellular organism requires that carefully orchestrated cell 46 division is integrated properly into different developmental processes. Cell division is 47 required not only to generate new cells that organize into tissues, but also to dictate 48 the size, position and timing of daughter cells that are generated. Several aspects of 49 cell division, including spindle orientation and division symmetry are well known 50 instruments of developmental programs (Siller and Doe, 2009). Roles for cytokinesis 51 in regulating developmental events are emerging, but are much less understood (Chen 52 et al., 2013; Herszterg et al., 2014;Li, 2007). Using advanced live imaging, we sought 53 to investigate cytokinesis in the well-defined divisions of the invariant C. elegans 54 embryo lineage, which has been completely described (Sulston et al., 1983). 55 Cytokinesis is the final step of cell division and is normally a constitutive 56 process during the exit from mitosis defined by discrete steps that occur during "C  elucidated, it is generally assumed that these events occur through a standard, well-77 defined series of ordered events. 78 A more clearly defined function for the midbody has been uncovered in cells 112 that undergo polarization events after the completion of cytokinesis. For example, 113 Madin-Darby canine kidney (MDCK) cells can establish apical basal polarity and 114 organize into a simple epithelial lumen structure (Reinsch and Karsenti, 1994). Apical 115 membrane markers are first delivered to the midbody during cytokinesis, establishing 116 an apical membrane at the interface between the first two daughter cells (Schluter et

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In order to further investigate patterns of cytokinesis during development, we 132 examined the invariant C. elegans lineage. We find that cytokinesis follows a lineage 133 specific pattern and that furrow symmetry and midbody inheritance is highly 134 reproducible. During morphogenesis, we observe striking midbody migration events 135 in the developing digestive and sensory tissues in C. elegans, likely before abscission. 136 Interestingly, AIR-2 migrates with midbodies and remains at several apical surfaces  Together, our results reveal that coordinated alterations in cytokinesis regulation, 143 particularly with regards to the midbody, are critical for proper animal development.

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Cytokinesis in the first two mitotic divisions: asymmetric midbody inheritance 146 We sought to systematically examine cytokinesis using lattice light sheet and 147 spinning disc confocal microscopy during the stereotypical divisions of the C. elegans 148 embryo, which has been extensively studied primarily in the first cell division due to 149 its size and ease of access. The first division of the P0 cell generates the larger 150 anterior daughter AB and the posterior daughter P1 (Fig. 1 A). We observed different 151 components that allow us to evaluate specific aspects of the cytokinetic apparatus 152 including the central spindle, the cytokinetic furrow and the midbody. We also chose 153 midbody markers that localize to the flank and ring sub-structures of the midbody 154 (Green et al., 2012). To observe the midbody flank region, we imaged the Aurora B 155 kinase, AIR-2, microtubules, and the membrane trafficking regulator RAB-11 ( Fig. 1,   156 3, and Video S1). We also imaged midbody ring markers including the non-muscle 157 myosin NMY-2 and the centralspindlin component ZEN-4 ( Fig. 1 G-P and Video S1).

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While the first mitotic furrow shows some variable asymmetry as previously  ). On average, the furrow symmetry parameter is 1.7 in the first division, while 181 the AB furrow is 21.6 and the P1 furrow is 16.1, indicating highly asymmetric 182 furrows in the second divisions ( Fig. 1 V, X). The central spindle is swept from the 183 middle of the AB cell into contact with EMS during furrow ingression ( Fig. 1 E,   184 Video S1). AIR-2 localizes to the central spindle, then the midbody flank and remains 185 associated with the midbody remnant after it is engulfed ( Fig. 1 D-  Therefore, all midbody markers examined behave as expected, localizing to the 192 midbody, which is internalized after abscission is completed.  polarization and subsequently organize into a tube (Leung et al., 1999). Our 216 observations demonstrate that these cells are performing cytokinesis as they undergo 217 polarization, which to our knowledge has not been previously reported (Fig. 2 A). The 218 E8 cells undergo relatively symmetrical furrowing that produces a centrally placed

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Next, we noticed that not all midbody components are internalized the end of 247 cytokinesis and some remain on the apical surface. In contrast to the midbody ring 248 components, AIR-2 persists at the apical midline well after the time that ring 249 components are internalized and polarization is complete (Fig. 2 D Video S2-4), co-localizing with the apical polarity marker PAR-6 ( Fig S2 F-H).

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Endogenous AIR-2 can also be observed at the apical midline by immunofluorescence  The air-2 (or207ts) embryos have only 53.6% (37/69) hatching even when left at 309 15 °C through hatching, indicating that this mutant is sick even at permissive 310 temperature, while wild-type N2, zen-4 (or153ts) and spd-1 (oj5ts) embryos are 100% 311 viable when kept at 15 °C (Table 1). Embryos shifted to 26 °C after 4.5 hours at 15 °C 312 (corresponding to late E4 to early E8 stages) showed significantly increased lethality 313 in both air-2(or207ts) and zen-4 (or153ts), but not spd-1 (oj5ts) (Table 1), which 314 correlates with the amount of cytokinesis failure observed. The few animals that were 315 able to hatch in air-2(or207ts) and zen-4 (or153ts) mutants had severe morphogenesis 316 defects (data not shown). Mutant embryos shifted after the completion of all the 317 developmental divisions at the comma to 1.5-fold stage were largely rescued for 318 lethality and hatched at a rate similar to permissive temperature (Table 1). Therefore, 319 these results are consistent with the hypothesis that cytokinesis is essential for the 320 final stages of embryonic development during morphogenesis.  was also observed in air-2 (or207ts) embryos, which were shifted at E4-E8 for 4.5-5 336 hours until the comma stage, indicating that these defects are not resolved later in 337 development ( Fig. S3 B, D). Furthermore, the intestine was highly mispositioned  despite having no lethality (Table 1) and minimal cytokinesis failures. Therefore, we 348 conclude that proper execution of cytokinesis is required for normal lumen formation 349 in the gut.

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Cytokinesis in the pharynx: apical midbody migration and AIR-2 accumulation 352 We also noticed migration of the midbody and accumulation of midbody form of cytokinesis just before they undergo dendrite morphogenesis (Fig. 6 A).

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These cells undergo a symmetrical furrowing event before midbodies form centrally 402 between the daughter cells ( Fig. 6 B and Video S8-9). A group of at least 6 daughter 403 cell pairs divide initially forming multiple midbodies as observed with both confocal 404 and lattice light sheet imaging (Fig. 6 C and Video S8-9). These midbodies migrate 405 together into a cluster over a 60-minute time window (Fig. 6 D). AIR-2::GFP, RAB-406 11 and tubulin persist in these clusters (Fig. 6 D, Fig. S5 A-F and Video S8-9), while 407 ZEN-4::GFP rapidly disappears during the midbody clustering process (Fig. 6 E and   408 Video S8). Endogenous AIR-2 can be observed in these lateral apical clusters (Fig. S1 409 K-M). We observe PAR-6 at the tip of the sensilla cluster, indicating that it is the 410 apical surface of these cells, which accumulates γ-tubulin::GFP similar to the pharynx 411 and gut (Fig. 6 G, Fig. S5 G-L). In contrast to ZEN-4::GFP, NMY-2::GFP migrates 412 with the midbody to the cluster and persists at the very tip of the dendrites (Fig. 6 F   413 and Video S8). To our knowledge, this is the first detailed examination of the division 414 and initial steps of organization of these neuronal cell precursors. normal as expected (Fig. 7 A). In the air-2 (or207ts) mutant, we observed numerous 445 defects in the subset of surviving embryos that did not fail to hatch and became L1 446 larvae (Fig. 7 B-E). Animals with no observed DiI staining were more common under 447 longer inactivating conditions (Table 2). All zen-4 (or153ts) fail to hatch when shifted 448 during E4-E8, preventing analysis of DiI staining (Table 2). When shifted from the E8 449 stage, the few surviving zen-4 (or153ts) larvae show severe DiI staining defects, 450 which was dramatically reduced if embryos were shifted after the final divisions at the 451 comma-1.5 fold stage (Fig. 7 F, Table 2). The spd-1 (oj5ts) animals still had weak 452 defects revealed by DiI staining despite having minimal cytokinesis failures, but never 453 showed a complete lack of staining (Fig. 7I, Table 2). These data are consistent with    The coordinated, directed movement of the midbody we observe in several 505 tissues represents a new phenomenon during cytokinesis. Our data also suggest that 506 abscission has not taken place before the midbody migrates in the intestine. This  Delineating the precise relationship between these two organelles and deciphering 588 how cytokinesis contributes to proper cellular reorganization during morphogenesis 589 will be a major focus of future studies.