Progressive Loss of Racgap1/Ogre Activity Has Sequential Effects on Cytokinesis and Zebraﬁsh Development

Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Developmental Biology

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Original research article Progressive loss of RacGAP1/ogre activity has sequential effects on cytokinesis and zebraﬁsh development

Rachel M. Warga n, April Wicklund, Sarah E. Webster, Donald A. Kane

Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008, USA article info abstract

Article history: RacGAP1 is one of the two components of the centralspindlin complex essential for orchestrating cy- Received 2 April 2016 tokinesis in all animal cells. We report here that the early arrest mutant ogre is a maternal and zygotic Received in revised form loss of function mutation in the zebraﬁsh homolog of racgap1. Like the other model organisms in which 27 May 2016 racgap1 is mutated, cells in the mutant stop dividing. In vivo cell recordings reveal that gradual loss of Accepted 16 June 2016 wild-type RacGAP1 leads progressively from a failure of abscission, then to cleavage furrow ingression, and ﬁnally complete absence of furrow formation. Despite the lack of cytokinesis, gross patterning occurs Keywords: overtly normally in ogre mutants and cells continue to cycle slowly, some even attaining four or eight Racgap1 nuclei. Many multinucleate cells differentiate and survive, but the majority of cells enter apoptosis that Cytokinesis we demonstrate is due to cumulative rounds of defective cytokinesis. Investigation of the cells that Cell cycle differentiate in the mutant indicate that RacGAP1 is also needed for long-term survival of motoneurons Motoneurons Microtubules and the cytoskeletal organization of sensory axons. We conclude that while RacGAP1 function is crucial Cytoskeleton for cytokinesis and its activity at different levels controls different aspects of cytokinesis, these defects have occluded other critical roles of this interesting protein. & 2016 Elsevier Inc. All rights reserved.

1. Introduction protein (GAP) RacGAP1, centralspindlin is only active as a complex, not as its individual components (Mishima et al., 2002; Pavicic- Cytokinesis is the final step of mitosis, cleaving the cytoplasm Kaltenbrunner et al., 2007). Moreover, the complexes must also of the dividing cell into two separate daughter cells. In animal self-associate into higher order clusters before concentrating on cells, cytokinesis begins with assembly of the central spindle, a the spindle midzone microtubules (Basant et al., 2015; Hutterer structure composed of bundled antiparallel microtubules, and a et al., 2009). In this paper we focus on the RacGAP1 subunit of complex of proteins known as centralspindlin. Once assembled at centralspindlin (known also as mgcRacGAP in mammals, Cyk4 in the midzone, the central spindle sets up a signaling center at the nematodes, and RacGAP50C in fruit flies). equator of the cell for positioning, assembly, and contraction of an Much of our knowledge concerning the role of RacGAP1 during actomyosin ring that drives cleavage furrow ingression. By the end cytokinesis has been gleaned from studies in early cleavage stage of furrow ingression the central spindle has compacted into a Caenorhabditis elegans embryos or cultured Drosophila and human structure known as the midbody: here it plays both structural and cells. Because loss of function mutations in nematodes and fruit fl regulatory roles in coordinating abscission and ending cytoplasmic ies are embryonic lethals (Jantsch-Plunger et al., 2000; Jones and continuity between daughter cells. In some animals and during Bejsovec, 2005; Unhavaithaya et al., 2013; Zavortink et al., 2005), less is known about other roles RacGAP1 may have. Nevertheless, very early development this may not occur until well into the next there is evidence that during polarization of the arcade cell epi- cell cycle (reviewed in: Agromayor and Martin-Serrano (2013), thelium in C. elegans, myotube extension and neurite outgrowth in Douglas and Mishima (2010), Glotzer (2013), Green et al. (2012), Drosophila as well as neural migration in mammals, RacGAP1 Schiel and Prekeris (2013), White and Glotzer (2012)). Mechan- (together with MKLP1) may have organizing properties of the istically it seems centralspindlin controls the myriad functions of cytoskeletal machinery (del Castillo et al., 2015; Falnikar et al., the central spindle including its own assembly (Pavicic-Kalten- 2013; Guerin and Kramer, 2009; Lores et al., 2014; Portereiko et al., brunner et al., 2007). Comprised of two proteins, the mitotic ki- 2004). Furthermore, there is mounting evidence that GAPs in nesin-like protein MKLP1 and the Rho family GTPase-activating general are critical players in neuronal development and survival making them underlying factors in many neurodegenerative dis- n Corresponding author. orders (reviewed in: Bai et al. (2015), Stankiewicz and Linseman E-mail address: [email protected] (R.M. Warga). (2014)). http://dx.doi.org/10.1016/j.ydbio.2016.06.021 0012-1606/& 2016 Elsevier Inc. All rights reserved.

Composed of multiple domains, RacGAP1 is a Swiss Army knife polymorphic WIK (L11) strain of wild-type fish for mapping by half of proteins. First, RacGAP1 contains a conventional RhoGAP. This tetrad analysis (Johnson et al., 1995)usingsingle-strandedpoly- domain inactivates the small GTPase Rac1 at the cell equator, ne- morphism of closely linked microsatellite markers (Knapik et al., 1998) cessary for successful furrow ingression (Bastos et al., 2012; Can- as in McFarland et al. (2005).Thismappedogre to one arm of Chro- man et al., 2008; D’Avino et al., 2004; Toure et al., 1998). Second, mosome 23. Finer resolution on the Sanger map was further obtained RacGAP1 provides specific binding sites for a number of key effec- using a haploid panel, which identified five potential candidate genes tors of cytokinesis and abscission. Notably, ECT2 (Burkard et al., in a 6.2 Mb interval. To identify which gene was ogre we used RT-PCR, 2009; Kim et al., 2014; Somers and Saint, 2003; Wolfe et al., 2009; this revealed a T to A transversion in racgap1 at nucleotide 1173 Zavortink et al., 2005), the guanine nucleotide exchange factor changing the reading frame from a cysteine to a stop. The mutation (GEF) needed to activate the small GTPase RhoA (Prokopenko et al., was confirmed by further sequence analysis using genomic DNA iso- 1999; Su et al., 2011; Yüce et al., 2005), which controls formation lated from individual mutant and wild-type embryos. and contraction of the actomyosin ring (Matsumura, 2005; Otomo et al., 2005; Piekny and Glotzer, 2008). Finally, RacGAP1 possesses 2.3. Antisense morpholino and mRNA injections an evolutionary conserved atypical C1 domain promoting protein- lipid interactions (Colon-Gonzalez and Kazanietz, 2006), a domain Morpholinos (Gene Tools, LCC) and mRNA were injected into 1- essential for linking the central spindle to the plasma membrane to 4-cell stage embryos. 4 nanograms of the exon8-intron8 splice and successful abscission (Lekomtsev et al., 2012). blocking morpholino (5′-AGGGAAATAATCTTACCAGTCTTCC-3′)was Here we describe ogre, a loss of function racgap1 mutation in injected per embryo to recapitulate the ogre mutant phenotype the zebrafish. Like the other model organisms in which racgap1 is and 5 nanograms of the racgap1 mRNA was injected per embryo to mutated, we find that cells in the ogre mutant become binucleate. rescue the ogre mutant phenotype. The racgap1 coding sequence However, this does not occur until gastrulation, long after onset of was purchased from (Open Biosystems) and transcribed with the zygotic transcription (Kane et al., 1996b), consistent with the idea SP6 mMessage mMachine (Ambion). that maternal stores of cell cycle gene products regulate cytokinesis during the first 15 or so cell cycles of zebrafish (Kane and 2.4. Lineage tracing Kimmel, 1993). Sequencing of the mutant allele revealed a nonsense mutation in the C1 domain, just upstream of the GAP do- Cells were labeled at the 1000–2000-cell stage using protocols main. By monitoring cytokinesis in vivo we show that abscission previously described in Warga et al. (2013) and Warga and Nüs- rather than cleavage furrow ingression is most sensitive to levels slein-Volhard (1999) with the following modification: embryos of RacGAP1 activity. However, once maternal racgap1 transcripts were injected with a 5% solution of neutral rhodamine-dextran become depleted, cleavage furrow ingression is compromised. (10,000 MW; ThermoFisher Scientific, formerly Molecular Probes). Notably, we also find a weak dominant maternal effect. Cells lacking zygotic RacGAP1 continue to re-enter the cell cycle after 2.5. Time-lapse analysis becoming binucleate albeit more slowly than wild type, however if cells are prevented from becoming quadnucleate apoptosis is rare. Embryos were mounted and recorded in multi-plane as pre- We also show that neural development proceeds in the absence of viously described in Warga and Kane (2003) except for experiments RacGAP1, even if cells are multinucleate, but primary motoneurons with the Dual FUCCI transgene or lineage tracer where we used a in particular fail to survive and sensory neurons exhibit cytoske- Nikon C2 confocal microscope. For embryos labeled with lineage letal defects. Our studies indicate that while RacGAP1 function is tracer cells were recorded every 3 min from sphere until early more crucial for the final stages of cytokinesis, it is also critical for gastrulation. For Dual FUCCI embryos, cells were recorded every some aspects of post mitotic neural development and survival. 5 min from midgastrulation until early somite stages. After each time-lapse, individual embryos were unmounted and allowed to develop to determine its phenotype (mutant or wild type). Re- 2. Material and methods cordings were analyzed using Cytos Software as in Warga and Kane (2007) or NIS elements viewer (Nikon). Images were imported into 2.1. Zebrafish strains Adobe Photoshop and pseudo-colored to aid in presentation.

All experiments except antisense morpholino injections were 2.6. Cell dissociation and percent of multinucleate cells performed in embryos derived from crosses of identiﬁed ogreta52a heterozygotes. For following the cell cycle, the dual FUCCI trans- Individual embryos were digested from 5 to 8 min (depending genic line (Bouldin and Kimelman, 2014) was also bred into the on stage) as in Riley et al. (2010) and resuspended in 1-part 4% orgre mutant background. Morpholino injections were performed paraformaldehyde containing DAPI (0.1 mg/ml) and 4-parts Holt- in embryos derived from wild type, the hrpti245 allele of Emi1 freter's buffer. Dissociated cells were immediately mounted on a (Riley et al., 2010) or the zomta94 allele of Cdc20 (D.A.K. un- hemocytometer and inspected at 400x magniﬁcation to determine published). The ogre, hrp and zom mutant phenotypes are only the number of nuclei for each cell. Typically, 100–300 cells were visible when homozygous and segregate 1:3 as a normal Mende- counted per embryo. Statistics were done using contingency tables lian recessive trait. Because there is no discernable heterozygous of the total or partial results from individual embryos using phenotype, throughout the paper when we refer to wild-type Pearson’s chi-squared test (without Yate's correction) and calcu- siblings (the remaining three quarters of the progeny) we are al- lating probabilities from the binomial distribution. Often these ways referring to their phenotype and not their genotype (which results were crosschecked using a simple Wilcoxon rank sum. could be either homozygous for the wild-type allele or heterozygous for the mutant allele). 2.7. In situ hybridization, antibody, acridine orange, and DAPI staining 2.2. Mapping and genotypic characterization Whole-mount RNA in situ hybridization was carried out using The ogreta52a allele, recovered in a large-scale ENU mutagenesis digoxigenin-labeled riboprobes following the protocol in Thisse screen (Haffter et al., 1996), was initially outcrossed to the and Thisse (2008).

Whole-mount antibody staining was carried out using an anti- and crushing gently. This rendered the embryo a loose association active Caspase 3 antibody (BD Biosciences, 1:200), an anti-Acety- of 2–3 cell layers, greatly increasing optical clarity of DAPI-stained lated Tubulin antibody (Sigma, 1:2000) or an anti-Phospho-His- nuclei. Photographs were taken immediately at 400 Â on a Zeiss tone H3 antibody (Santa Cruz Biotechnology, Inc., 1:1000) and Axioskop 2 of typical cells. detected with an AP-conjugated or peroxidase-conjugated sec- ondary antibody (Santa Cruz Biotechnology, Inc. 1:1000). For vi- 3. Results sualization of microtubules at 1000 Â magnification with anti- Acetylated Tubulin, we used an Alexa Fluor 488-conjugated sec- 3.1. Cells in the ogre mutant undergo apoptosis ondary antibody (Invitrogen 1:500). Acridine orange staining was carried out by placing live de- Identified in the Tübingen zebrafish screen for early morpho- À chorionated embryos in 10 5 M acridine orange with 1% DMSO in genetic mutants, the mutant ogre was characterized as an early the dark for 30 min, washing brieflyinfish H2O several times and arrest phenotype with massive cell death and is represented by a examining immediately with fluorescence. single recessive allele ta52a (Kane et al., 1996b). The earliest visible DAPI staining was obtained by placing an embryo between phenotype is a shorter body once somites begin to form (11 h), and coverslips in 4% paraformaldehyde containing DAPI (0.1 mg/ml) by 6-somites (12 h) the eye placode is smaller than normal and

Fig. 1. The ogre mutant phenotype. (A and B) Live embryos; (C) after acridine orange staining to show cell death; and (D) after DAPI-squash to show nuclei. (E–H) anti-Active Caspase 3 staining showing progression of apoptosis; and with dlA expression (I) marking neural tissue in the brain and spinal cord, with myoD expression (K) marking somite muscle in the trunk, and with gata1a expression (M) marking blood in the posterior blood island. (J, L, N) Are high magniﬁcation cross-sections through the areas indicated with lines and tissues are circled while arrows indicate apoptotic cells; abbreviations: nt, neural tissue; nc, notochord. (O–Q) Expression of: (O) tal1 mRNA marking blood cells (arrows) and (P) Acetylated Tubulin marking trigeminal (trg) and Rohon-Beard sensory (rb) neurons (arrows). (Q) High magniﬁcation of mutant axons, showing abnormal varicosities (arrows).

Fig. 2. ogre is a mutation in racgap1. (A) Genetic map of chromosome 23 showing number of recombinants and calculated distance for microsatellite markers and genes. (B) Schematic of the racgap1 cDNA showing location of termination codon in exon 10 and (C) sequence trace data. (D) Schematic of the RacGAP1 protein. MKLP1 binding region; CC, coiled-coiled domain; ECT2/FIP3 binding region; C1, atypical C1 domain; GAP, GTPase-activating domain. (E–H) Expression of racgap1 mRNA: (E) maternal expression at 1 h; and (F–H) zygotic expression at: (F) 7 h; (G) 18 h; and (H) 24 h. (I–J) Anti-sense knockdown of racgap1 mRNA by morpholino (MO) phenocopies ogre; left side: embryo, right side: DAPI-stained nuclei. (K-P) racgap1 mRNA rescues the ogre mutant phenotype. (K) racgap1 injected and (L) kaede control embryos. (M) Quantification of multinucleate cells at 10-somites. Differences between kaede and racgap1 injected mutant embryos are significantly different (po0.01, Wilcoxon rank sum); racgap1 injected wild type and mutant embryos are also significantly different (po0.01, Wilcoxon rank sum). (N–P) dlA expression in neuroblasts: (N) racgap1 injected and (O) kaede control embryos and (P) their average number in ogre mutants.

Please cite this article as: Warga, R.M., et al., Progressive loss of RacGAP1/ogre activity has sequential effects on cytokinesis and zebrafish development. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.06.021i R.M. Warga et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5 apoptosis occurs in a rostral-caudal gradient not only in nervous transcripts no longer compensate for zygotic transcripts (Kane tissue but also in mesodermal tissues such as muscle and blood. In et al., 1996a, 1996b). Beginning after the onset of gastrulation (6 h), summary, it seems cell death occurs in any tissue with actively 3 h after the onset of zygotic transcription (Kane and Kimmel, dividing cells. 1993) and when the embryo has segregated into three spatially Previously we reported that cells in the ogre mutant exhibit distinct cellular domains (Kane et al., 1992), we found that the double nuclei as somites begin to form (Kane et al., 1996b). Con- behavior of cells dividing in the deep cell domain of the blas- sistent with the idea that defective cytokinesis may be responsible toderm (the embryo proper) was markedly different between for cell death, a high proportion of mutant cells are binucleate by mutant and wild type. Upon cleavage furrow ingression daughter 9-somites (Fig. 1D). Regardless, many cell types develop in ogre cells in the mutant separated from each other as in wild type but mutants including those that maintain actively dividing cell po- then slowly coalesced back together as if they were unable to pulations such as blood and neurons (Fig. 1O and P). However, physically sever the cytoplasmic bridge between each other their differentiated progeny at 24 h are far fewer in number and (compare Fig. 3B to A). This cell behavior persisted until 75% epi- often larger in size compared to wild type (arrows in O and P). boly (8 h; Fig. 3C, blue cell) whereupon a more abbreviated version Moreover, the axons of sensory neurons are more disorganized of cytokinesis appeared. Here a cleavage furrow formed, but it and appear to overgrow as well as exhibit abnormal swellings rapidly regressed immediately coalescing the future daughter cells along their length (Fig. 1P and Q). back together (Fig. 3C, purple cell). Both these cell behaviors are illustrated in Fig. 3E. By 90% epiboly (9 h; Fig. 3D), some binucleate 3.2. ogre is a mutation in racgap1 cells were now beginning to divide. Here cells exhibited almost no cleavage furrow, but underwent karyokinesis. As nuclei re- We mapped ogre via half-tetrad analysis to chromosome 23 and appeared they were often unequal in size suggesting possible using a haploid mapping panel fine-mapped ogre to the interval defects in aneuploidy. Notably, cells that failed at furrow ingres- between the simple sequence length polymorphisms z5141 and sion also no longer rounded up during mitosis, instead, they z31657 (Fig. 2A). This area was further refined using the nearby wrinkled exhibiting tiny projections all over their surface (Fig. 3C linked gene grasp1 (grp1)thathadasizepolymorphismthatcould and D). A similar phenotype is observed in cultured human cells be visualized on an agarose gel, placing ogre near the zebrafish expressing a mutated GAP domain of RacGAP1, a defect attributed homolog of racgap1. Sequencing of the mutant coding sequence to improper cell adhesion at the cleavage plane of the dividing cell revealed a nonsense mutation in exon 10 (Fig. 2B and C) that occurs (Bastos et al., 2012). We conclude that falling levels of RacGAP1 within the predicted atypical C1 domain and before the catalytic first compromise abscission and then cytokinesis entirely. GAP domain (Fig. 2D), domains that are highly critical for cytokinesis A related cellular behavior was observed in the extraembryonic (Basant et al., 2015; Bastos et al., 2012; Canman et al., 2008; Le- domain of the blastoderm, where cells normally cycle more slowly komtsev et al., 2012; Loria et al., 2012). In situ analysis showed that (Kane et al., 1992). However, rather than cytokinesis failing, it was zebrafish racgap1 mRNA is expressed maternally (Fig. 2E), consistent unusually delayed. Starting around the onset of epiboly (4.3 h), with the idea that maternal transcripts must be depleted before cell some enveloping layer cells entering mitosis completed nuclear division defects appear (Kane et al., 1996b), as well as zygotically division, but like deep cells later, did not complete cytoplasmic (Fig. 2F–H). In the gastrula, expression was ubiquitous, but by 1 day division and fused back together (Fig. 3F). Usually, before gas- expression was highest in the nervous system, particularly the brain. trulation, most of these binucleate cells rounded up and exhibited Investigation of ogre mutants revealed that racgap1 mRNA was violent protrusive activity (Fig. 3F, panel 1:17:00) that may be akin mostly depleted by 18-somites (18 h; Fig. 2G′) and not detectable by to the cell projections seen later all over dividing deep cells, before 1day(Fig. 2H′) in keeping with depletion of mRNA transcripts that subsequently dividing into two mononucleate daughter cells that are subjected to nonsense-mediated degradation. possibly are tetraploid rather than diploid. Though transient, this To confirm the ogre phenotype results from a mutation in phenotype was 100% penetrant regardless of zygotic genotype, racgap1, we injected a splice-blocking morpholino against racgap1 demonstrating it to be a maternal dominant phenotype. Never- transcripts. Antisense inhibition completely recapitulated the theless, during early gastrulation, this marked behavior dis- morphological and cytological defects of the ogre mutant (Fig. 2I appeared in all but the homozygous mutant where a cell cycle and J). Next we injected in vitro synthesized mRNA for racgap1 or a later cytokinesis stopped altogether in enveloping layer cells. control mRNA encoding the photoconvertible fluorescent protein Hence, rising zygotic levels of RacGAP1 return cytokinesis to Kaede (Hatta et al., 2006). This demonstrated that wild-type rac- normal. gap1 mRNA delayed onset of the ogre phenotype (Fig. 2K and L), partially rescued the cytokinesis defect (Fig. 2M), increased the 3.4. Mutant cells reenter the cell cycle after becoming binucleate number of neural cells (Fig. 2N–P) and even rescued motor function in mutant embryos (Supplementary Fig. 1). We conclude from To characterize in more detail mutant cytokinesis, we dis- these experiments that ogre is a null allele and a complete loss of sociated embryos derived from heterozygous mutant carriers at zygotic RacGAP1 function. We also validate that the racgap1 different stages of development. Because cell number in the mu- morpholino completely replicates the ogre mutant phenotype. tant does not increase due to defective cytokinesis, this allowed us to calculate the percent of cells that were mononucleate, bi- 3.3. Mutant cells exhibit defects in abscission before defects in nucleate or quadnucleate for a single embryo (Fig. 4A–C and cleavage furrow ingression Supplementary Fig. 2) and thus illustrate how many times a cell attempted to divide. Octonucleate cells were also observed, but RacGAP1 has a well-studied role as a component of the cen- only rarely. In agreement with above, by 70% epiboly (7 h) and tralspindlin complex which itself has several critical functions well before we could identify mutants morphologically, a quarter during cytokinesis including positioning of the cleavage furrow, of the dissociated embryos exhibited from 11% to 23% binucleate ingression of the cleavage furrow and abscission. To better un- cells (Supplementary Fig. 2A). The average number of binucleate derstand how cells in the mutant become multinucleate we video cells in these embryos (14%), contrasted sharply to the remainder time-lapsed embryos from 4 to 10 h of development. We chose of the embryos where few cells on the average were binucleate this period because mutants that affect the cell cycle are likely to (3%) consistent with this portion being phenotypically wild type exhibit their phenotypes during this time once maternal (Fig. 4D) and likely reflecting normal cells caught in cytokinesis.

Fig. 3. ogre mutants have both maternal and zygotic cytokinesis defects. (A–D) DIC bright ﬁeld recordings of the zygotic cytokinesis defect. Panels are selected frames from two simultaneously recorded video time-lapses. (A) Wild type and (B) mutant sibling at the onset of gastrulation (6 h), initially mononucleate cells (blue) round up, divide and separate before fusing back together in the mutant. (C) Mid gastrulation (8 h), mutant cells attempting to divide round up and exhibit tiny projections over their cell surface, some cells (violet) fail to complete furrow ingression whereas other cells (blue) do. (D) Late gastrulation (9 h), binucleate mutant cells (green) also exhibit projections as they round up only no cleavage furrow forms. (E) Schematic of cytokinesis defects. Early gastrulation, daughter cells separate, but fuse back together. Late gastrulation, the furrow regresses and cells fuse back together. (F) Fluorescent recording of the transient maternal cytokinesis defect. At onset of epiboly (4.3 h), an enveloping layer cell (labeled with lineage tracer) in a phenotypic wild-type embryo completes furrow ingression before fusing back together. An hour later the binucleate cell rounds up and exhibits violent protrusive activity before it successfully divides into two characteristic enveloping layer cells.

From tailbud (10 h) to the 4-somite stage (11.3 h), the average attrition of quadnucleate cells between 4- and 10-somites (14 h) is number of quadnucleate cells rose from 4% to 18% in the portion of due to apoptosis and that as a result mononucleate cells become dissociated embryos we deemed mutant based upon the fre- more highly represented. Later, from 10- to 20-somites (19 h) the quency of multiple nuclei in our counts (Fig. 4D and Supplemen- average number of quadnucleate cells rose again (28%) and the tary Fig. 2B and C). Simultaneously, the average number of bi- average number of mononucleate cells fell once more (38%), while nucleate cells in these embryos rose to 62% while the average during this time the average number of binucleate cells remained number of mononucleate cells fell to 20%. Hence by early somite more or less the same (34%; Fig. 4D). Therefore it seems that stages much of the embryo is multinucleate and many cells have most cells reenter the cell cycle and attempt to divide at least once attempted to divide at least once after cytokinesis is blocked. After after cytokinesis is blocked. As with our observations earlier, in the 4-somite stage, when ogre mutants can be unambiguously embryos that were phenotypically wild type, cells were almost identiﬁed before dissociating (Supplementary Fig. 2D and E), the exclusively mononucleate for all time periods (Fig. 4D). average number of quadnucleate cells fell to 9% and the average We veriﬁed that cells reenter the cell cycle and are mitotically number of mononucleate cells rose to 55% (Fig. 4D). As this cor- active in ogre mutants using Phospho-Histone H3 staining, which relates to a period when apoptosis is marked (Fig. 1F–H) and there labels the chromosomes from prophase to early anaphase (Hend- is no reciprocal increase in octonucleate cells, we conclude that zel et al., 1997). There was little difference in the apparent number

Fig. 4. Mitosis continues after cytokinesis fails. (A–D) Dissociated cells. (A, B) Low magnification and (C) high magnification showing range of nuclear phenotypes. (D) Quantification of multinucleate cells. At 7 h, the proportion of cells with double nuclei ranked embryos; mutant embryos were then inferred by the simple Mendelian segregation of embryos with the highest deviation from wild type patterns. At 10 and 11.3 h, mutant embryos were inferred based on cell counts deviating from the wild type proportions. At 14 and 19 h, mutant embryos were identified morphologically before cell dissociations. The results of all of the embryos along with a partial statistical analysis are in Supplemental Fig. 2.(E–I) Multinucleate cells are mitotically active. (E–H) Phospho-Histone H3 staining and egr2b expression to visualize rhombomere 4, (E–G) are side views, insets in (E) are higher magnification of mitotic nuclei and (H) high magnification dorsal view of rhombomere 4. (I) Quantification of mitotic multinucleate cells at 10-somites in rhombomere 4; n indicates the number of embryos scored. Data are significantly different at the p{0.001 level (contingency table tested with Pearson's chi square). of mitotic cells between wild type and mutant at 4-somites already by 12-somites (15 h) mutant erythrocytes mostly had two (Fig. 4E); at this time mitotic mutant cells were either mono- or four nuclei (Fig. 5C). Consequently if mutant erythrocytes cycle nucleate or binucleate (inset). However, by 10-somites ogre mu- as often as their wild-type counterparts, changes in their number tants had notably fewer mitotic cells compared to wild-type em- should echo those in wild type only in reverse, due to cells dying bryos (Fig. 4F). This was confirmed by counting the number of cells once they have four nuclei as we have already demonstrated. in rhombomere 4, outlined by expression of the zinc-finger tran- As expected, in wild-type embryos, erythrocytes progressively scription factor egr2b (Oxtoby and Jowett, 1993). On the average increased in number every 5–6 h from 316 721 cells at 15 h to there were 16.971.0 mitotic cells in wild type versus 4.170.3 920725 cells at 32 h (Fig. 5G). In ogre mutants, because most mitotic cells in mutants (Fig. 4H). By now the majority of mitotic cells already had at least two nuclei to start with, there were in- cells in mutants were binucleate (69%; Fig. 4I), but many were itially half as many erythrocytes than in wild type (158 721) and quadnucleate (21%), suggesting that perhaps they die because of a cell cycle later at 22-somites (20 h), their number was almost the entering mitosis. Not many mitotic cells were mononucleate (10%), same, indicating cells were not dying. Two cell cycles later (26 h), however because these cells did not divide earlier and waited till there were fewer erythrocytes (8776 cells), but a cell cycle after now to enter mitosis, likely explaining their reduction between 10- that (32 h) there was little change (Fig. 5G). Therefore between 15 and 20-somites (Fig. 4D), this suggests there may be a population and 32 h, mutant erythrocytes appear to cycle only once whereas of cells that have a very long cell cycle. At one day few mitotic cells wild-type erythrocytes appear to cycle mostly twice. In support of were apparent in ogre (Fig. 4G) and these were largely abnormal. this view, at 26 h when mutant erythrocytes begin to die a cell Hence cells in ogre mutants continue to cycle after cytokinesis is cycle after they should, they now possessed mostly four nuclei blocked, but once a cell has four nuclei it is detrimental and fur- (Fig. 5D). Other blood cells, macrophages and neutrophils, nor- ther attempts to divide are likely lethal. mally have a much longer cell cycle than erythrocytes, but still divide at least once during this time interval (Warga et al., 2009). 3.5. The cell cycle is longer in ogre mutant cells We counted these cells as well (Fig. 5G) using lcp1 (Fig. 5E), an actin binding protein expressed in differentiating macrophages Our proceeding study indicates that the cell cycle may be (Herbomel et al., 1999) and mpo (Fig. 5F), a peroxidase expressed longer in a population of mutant cells. It is well known that cells in differentiating neutrophils (Bennett et al., 2001; Lieschke et al., respond to cellular stress by delaying their cell cycle (reviewed in: 2001). In contrast to wild type, there was little change in their Clarke and Allan (2009), Rieder and Maiato (2004)) as might occur number over this time span in ogre. once RacGAP1 function is lost and cells have perhaps more than To investigate this further, we visualized the cell cycle in real one nucleus. Therefore to determine if the cell cycle is longer in time using the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI). the ogre mutant we counted the number of blood cells in fixed This is a two-color system that makes use of oscillating levels of embryos from 15 to 32 h of development at roughly 5 h intervals two key cell cycle proteins: Cdt1, which accumulates during G1 using gata1a (Fig. 5A), a transcription factor specific to ery- and Geminin, which accumulates during S, G2, and early M (re- throcytes (Detrich et al., 1995) and hbbe2 (Fig. 5B), a globin ex- viewed in: Caillat and Perrakis (2012), Zielke and Edgar (2015)). In pressed in differentiating erythrocytes (Brownlie et al., 2003). We zebrafish, a single transgene expresses a Cdt1-degron tagged with chose this method because erythrocytes normally divide once mCherry and a Geminin-degron tagged with Cerulean (Bouldin every 5–6 h during the first 36 h of development if cells have not and Kimelman, 2014) to report the different phases of the cell yet left the cell cycle (Warga et al., 2009) and this time because cycle as illustrated in Fig. 5H. Using this transgene we performed

Fig. 5. The cell cycle is longer in ogre. (A, B) Expression of: (A) gata1a and (B) hbbe2 in erythrocytes. (C, D) High magnification view of erythrocytes at 15 h (C) and at 26 h (D); numbers beside cells indicate their number of nuclei (dotted), not all nuclei are visible in the plane of focus, (E, F) Expression of: (E) lcp1 in macrophages and (F) mpx in neutrophils. (G) The average number of blood cells at different times of development. For erythrocytes we used gata1a expression at 15 and 20 h and hbbe2 expression at 26 and 32 h, for macrophages we used lcp1 expression and for neutrophils we used mpx expression, which comes up later. We examined 5 wild type and 5 mutant embryos per stage. (H) Schematic of the dual Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) with respect to the cell cycle. (I–K) Selected frames from three confocal time-lapses used to quantitate how long cells spent in late M and cytokinesis (when there is no signal) and the G1/S transition (when nuclei are pink). For each nucleus and its daughter nuclei, the stage of the cell cycle is indicated. Embryos (I) and (J) are siblings and were recorded simultaneously. Note that in mutant (J) cells take not only longer to transition through mitosis and G1/S, but that the Cdt1 degron takes longer than the Geminin degron to build expression in the G1/S transition. Note also that in mutant (K) the two daughter nuclei do not cycle together. (L) Quantification of G1/S transition and mitosis; n indicates the number of cells and in all but one case only one cell was used per embryo. Respectively, differences are significant at po0.005 and po0.002 (Wilcoxon Rank Sum). R.M. Warga et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 9 time-lapse confocal microscopy beginning at 75% epiboly (8 h) to apoptosis (Fig. 6B and C, compare row 1 and 2) suggesting that quantify the length of time mutant cells and their wild-type excess chromosomal content is not the reason ogre mutant cells counterparts spent in the G1/S transition (when cells are pink due die. Knockdown of RacGAP1 in the hrp mutant shows cells do not to the presence of both Geminin and Cdt1) and in late M and cy- need RacGAP1 to survive if they do not divide for while wild-type tokinesis (when no signal is seen because Geminin is degraded). siblings depleted of RacGAP1 display double nuclei and extensive Here we focused on enveloping layer cells because they do not apoptosis, hrp mutant embryos do not (Fig. 6D–F, compare row undergo as extensive morphogenetic movements as the deeper 1 and 2). Hence defective cytokinesis likely initiates apoptosis. cells of the embryo (Fig. 5I and J). Notably even though mutant To further test this hypothesis and exclude the possibility that cells had only one nucleus to start with, the average length of time the hrp mutation might be repressing apoptosis from happening, mutant cells spent in late M and cytokinesis was considerably we turned to another cell cycle mutant to block cytokinesis. For longer than wild-type cells and the same was true for the G1/S these experiments we used the mutant zombie (zom; Fig. 6A) transition (Fig. 5L). Thus lengthening of the cell cycle in ogre carrying a mutation in cdc20 (Kane et al., 1996b) and (D.A.K. un- mutants may have little to do with the number of nuclei and more published). Upon running out of maternal product around the end to do with absence of RacGAP1 itself. However, we also observed of gastrulation (10 h), cells in homozygous zom mutants arrest that after mitosis not all daughter nuclei cycled together (Fig. 5K), after the chromosomes condense in either prophase or metaphase not unusual for normal daughter cells (Kane et al., 1992; Kimmel (Fig. 6B), but like hrp, there is little apoptosis (Fig. 6C, compare row and Warga, 1986, 1987; Kimmel et al., 1994; Warga and Kimmel, 2 and 3). Knockdown of RacGAP1 in the zom mutant resulted in a 1990), but perhaps contributing to slowing of the cell cycle once more severe morphological phenotype (Fig. 6D) that exhibited a cells are multinucleate. These results and those above demonstrate range of nuclear phenotypes (Fig. 6E) as the zom mutant pheno- that different cellular domains, both embryonic (blood) and ex- type occurs later than the hrp mutant phenotype and RacGAP1 traembryonic (enveloping layer cells) cycle more slowly in ogre. depleted cells mostly do not arrest in mitosis until after becoming binucleate. Typically both nuclei were in some state of mitotic 3.6. Stopping cytokinesis prevents RacGAP1-dependent apoptosis arrest. Usually both were in prophase or in metaphase (red arrow heads), but infrequently one of the nuclei was in prophase and the Cells also respond to cellular stress by initiating a program of other in metaphase (red arrow) agreeing with our observation cell death through the process of apoptosis (reviewed in: Clarke above that not all daughter nuclei are in synchrony with one an- and Allan (2009), Fulda et al. (2010)). To determine if defective other (Fig. 5K). However, there were also cells where both nuclei cytokinesis is causing mutant cells to enter apoptosis we pre- had not yet arrested (yellow arrowhead) or only one (yellow ar- vented cell division from occurring in embryos injected with the row). Significantly, loss of zom function decreased RacGAP1-de- racgap1 morpholino (Fig. 6). Here we took advantage of a novel pendent apoptosis (Fig. 6F, compare row 1 and 3) demonstrating cell cycle mutant harpy (hrp; Fig. 6A), carrying a mutation in the that cells lacking RacGAP1 will not enter apoptosis, even with gene emi1 (Kane et al., 1996b; Rhodes et al., 2009; Riley et al., excess chromosomes and two nuclei, if cytokinesis is blocked. zom 2010; Zhang et al., 2008). Upon running out of maternal product however, was not as effective as hrp at preventing apoptosis around the onset of gastrulation (6 h), cells in the homozygous (Fig. 6F, compare row 2 and 3) likely because some binucleate cells mutant bypass mitosis completely and instead undergo repeated that were not arrested divided once more. In sum these results rounds of DNA synthesis. This phenotype results in super sized demonstrate that apoptosis is not due to lack of RacGAP1 per se, cells with large polyploid nuclei that mostly do not undergo but rather to defective cytokinesis that results when RacGAP1

Fig. 6. RacGAP1 depleted cells survive if cytokinesis is prevented. Row (1) phenotypic wild type, row (2) homozygous hrp mutants, and row (3) homozygous zom mutants. Row 1 and 2 are siblings. Column (A–C) uninjected embryos; column (D–F) morpholino (MO) knockdown embryos. (A, D) Live embryos, (B, E) DAPI stained nuclei: red arrowheads indicate sister nuclei where both nuclei are arrested in prophase; red arrow indicates two sister nuclei where one nuclei is in prophase and the other nuclei is in metaphase; yellow arrowheads indicate sister nuclei where both nuclei are in interphase; and yellow arrow indicates two sister nuclei where one nuclei is in interphase and the other nuclei is in prophase, and (C, F) anti-active Caspase 3 stained embryos.

Fig. 7. The ogre nervous system lacks later born neurons. (A, B) Expression of: (A) notch1b in proliferating cells and (B) dlA in neuroblasts. (C, D) High magnification view of neuroblasts visualized with dlA and DAPI. Numbers beside circled dlA-positive cells indicate their number of nuclei or if the cell was apoptotic (apop), white bar shows the boundary between somites 3 and 4. (E) Expression of islet1 in primary motoneurons (pmn) and Rohon-Beard sensory neurons (rb). Inset (E′) expression of col2a in floorplate neurons (flp) and hypochord cells (hc). (F) Expression of: pax2a and egr2b in CoSA interneurons (CoSA) and inset (F’) high magnification of CoSA interneurons. Numbers beside cells indicate their number of nuclei. (G) Quantification of multinucleate neuroblasts between somites 2 and 8; n indicates the number of embryos at different stages. Differences are significant compared between 14 h and 17 h (po0.001) and compared between 17 h and 21.5 h (po0.05; contingency tables/Pearson's chi square).

Please cite this article as: Warga, R.M., et al., Progressive loss of RacGAP1/ogre activity has sequential effects on cytokinesis and zebrafish development. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.06.021i R.M. Warga et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 11 their steady decline in number (Fig. 7B), there was an increasing (Fig. 8F), a phenotype also observed with possible presumptive tendency for specified neurons to appear apoptotic rather than presynaptic swellings that tended to be larger, more tangled and multinucleate after 10-somites (Fig. 7G) in keeping with the idea more frequent than normal (Fig. 8H and I). The large tangles of that not all neuroblasts have yet left the cell cycle and made the microtubules along peripheral axons likely explains the con- switch from proliferation to differentiation before expressing dlA spicuous swellings we observed at lower magnification (Fig. 1P (Appel et al., 2001). These likely are the cells that are dying. and Q) a phenotype that would be enhanced by using an enzy- To see what cells in the nervous system differentiate we ex- matic reaction. We conclude from these results that RacGAP1 is amined the expression of two post mitotic markers: islet1 (isl1) not only required for organizing the cytoskeleton in dividing cells and pax2a (Fig. 7E and F). Here we concentrated on the spinal cord but also for organizing the cytoskeleton in post mitotic neurons. because the brain of ogre mutants is almost nonexistent by one day (Fig. 7F; compare brain expression between mutant and wild type). In the spinal cord, the LIM/homeodomain transcription 4. Discussion factor isl1 is expressed in primary motor and sensory neurons at the time these cells differentiate (Appel et al., 1995; Inoue et al., Here we describe a zebrafish mutation in racgap1, one of two 1994; Korzh et al., 1993) while the paired-box homeodomain components of the centralspindlin complex essential for orches- transcription factor pax2a is expressed in CoSA interneurons trating cytokinesis in all animal cells. We show that with de- (Krauss et al., 1991; Mikkola et al., 1992). We found that at 12- creased maternal RacGAP1 there is a marked delay between nu- somites (15 h), the expression of isl1 and pax2a was remarkably clear division and cell division, whereas in the absence of zygotic similar between mutant and wild type. However at one day, their RacGAP1 abscission fails and then finally cleavage furrow ingres- expression indicated that only the earliest born neurons differ- sion. Cells that no longer complete cytokinesis continue to cycle, entiate in ogre mutants for no more Rohon-Beard sensory or CoSA albeit more slowly, and can become quad or even octonucleate. interneurons were visible than earlier, much like happens in other However, while many mutant cells differentiate containing mul- cell cycle mutants (Johnson et al., 2014; Riley et al., 2010; Song tiple nuclei—and survive—more often they enter apoptosis. By et al., 2004). Therefore early born neurons differentiate in ogre, preventing mutant cells from re-entering the cell cycle we de- even though many possess multiple nuclei (Fig. 7F′), but later born monstrate that apoptosis is not due to lack of RacGAP1 per se, but neurons such as those posterior in the embryo (Kimmel et al., rather cumulative rounds of defective cytokinesis. We also de- 1994) do not, in agreement with our results above that later spe- monstrate that RacGAP1 is not only required for cytokinesis, but cified neurons tend to die. Unexpectedly, primary motoneurons no also for survival of some post mitotic neurons as well as the cy- longer were apparent by one day (Fig. 7E). Thus they died some- toskeletal organization of axons. time after differentiating, even though adjacent floorplate neurons (identified by col2a1a expression) still were present (Fig. 7E′). As 4.1. ogre is a loss of function maternal zygotic mutation no other mutation in the cell cycle has a phenotype where cells differentiate and later die before the embryo (Johnson et al., 2014; We demonstrate by antisense knockdown and wild-type mRNA Riley et al., 2010; Song et al., 2004), this shows that some cell types rescue that the ogre mutant phenotype is unequivocally the result such as primary motoneurons must require RacGAP1 post mito- of a premature termination (nonsense) codon in exon 10 of rac- tically to survive. gap1. If translated, mutant mRNAs would produce a shorter protein having a truncated atypical C1 domain and no catalytic GAP 3.8. Microtubule organization is aberrant in mutant sensory axons domain—each highly critical for cytokinesis (Basant et al., 2015; Bastos et al., 2012; Canman et al., 2008; Lekomtsev et al., 2012; In Drosophila when either the fly homolog of RacGAP1 or Loria et al., 2012). However, in situ analysis shows that racgap1 MKLP1 is mutated or depleted (del Castillo et al., 2015; Goldstein transcripts are present in lesser amounts in mutant embryos and et al., 2005), axons overgrow. ogre mutants have a similar phe- eventually lost in keeping with these mRNAs being targeted for notype and their axons exhibit visible swellings along their length degradation through the process of nonsense-mediated mRNA (Fig. 1P and Q). To look at this morphology in more detail, we decay (reviewed in: Chang et al. (2007)). Therefore, these results examined the peripheral processes of trigeminal sensory and Ro- indicate that ogre is most probably a loss of function mutation. hon-Beard neurons using acetylated tubulin staining and fluor- In zebrafish, racgap1 mRNA is expressed maternally and zygo- escent microscopy. High magnification revealed that the micro- tically and like other model organisms where racgap1 is mutated, tubule organization in mutant axons was impaired, although this cells in homozygous ogre mutants become binucleate. Excepting phenotype was not completely penetrant possibly because these the dominant maternal phenotype which is transient, binucleate sensory neurons are among the earliest neurons to differentiate cells do not appear until long after the onset of zygotic tran- (9–10 h) and send out processes (4–5 h later; Lewis and Eisen, scription (Kane et al., 1996b), consistent with the idea maternal 2003; Metcalfe et al., 1990). Thus it is likely that in these cells stores of cell cycle gene products regulate cytokinesis during the maternal products have not yet been completely exhausted first 15 or so cell cycles of zebrafish (Kane and Kimmel, 1993). The (Fig. 2 G). transient maternal phenotype, where extraembryonic cells be- In wild type (Fig. 8A), peripheral axons of trigeminal and Ro- come binucleate before eventually dividing likely is due to half as hon-Beard neurons branch extensively over the lateral surface of much maternal wild-type mRNA. Hence there are distinct ma- the embryo (Metcalfe et al., 1990). When processes bifurcate the ternal and zygotic features to the cytokinesis defect. microtubules within a process defasciculate and fan out before bundling back together into one or the other branch (Fig. 8B). 4.2. RacGAP1 and cytokinesis Occasionally loops of microtubules also occur along processes (Fig. 8C) that resemble the architecture of a presynaptic varicosity Numerous studies have examined the role of centralspindlin—a (Chang and Balice-Gordon, 2000; Conde and Cáceres, 2009). In protein complex consisting of the Rho family GTPase-activating mutants (Fig. 8D and G), peripheral axons were more disorganized, protein RacGAP1 and its partner the kinesin MKLP1—for its var- frequently wandering back and forth, and their microtubule or- ious functions during cytokinesis including ingression of the ganization at branch points was rarely normal (Fig. 8E). More often cleavage furrow, formation of the midbody and abscission (re- at bifurcations the microtubules were tangled around each other viewed in: Davies and Canman (2012), Glotzer (2013), Green et al.

Fig. 8. Microtubules are disorganized in mutant axons. (A–I) 30 h embryos visualized with anti-Acetylated Tubulin. (A, D, G) Low magnification, showing pathfinding errors in trigeminal (trg) and Rohon-Beard (rb) peripheral axons; highlighted boxes indicate regions shown at higher magnification in panels (B, C, E, F, H, I). (B, C) Wild type: (B) a branch point where individual microtubules defasciculate and fan out and (C) a presumptive presynaptic swelling (arrow) where individual microtubules form a loop. (E, F) ogre: (E) a normal looking branch point and (F) a branch point where microtubules tangle. (H, I) ogre: (H) three presumptive presynaptic swellings where one looks normal (arrow) and two not normal (indicated with asterisk) due to them being immediately adjacent to each other and appearing swollen (the one on the left may also be instead a region where individual microtubules abnormally defasciculate from one another and tangle), and (I) two presumptive presynaptic swellings along the same neurite (the picture is cropped to show both) where instead of forming loops the microtubules form large irregular tangles (asterisks). Note how in general mutant presumptive presynaptic swellings are larger than wild type.

(2012), Schiel and Prekeris (2013), White and Glotzer (2012)). cytokinesis in an intact vertebrate embryo carrying a presumptive However few of these studies have used live imaging analysis to null mutation show cytokinesis decays over time due to the characterize the cytokinesis defects in an intact embryo and to elimination of maternal racgap1 transcripts. Cytokinesis defects date the only animal where this has been done extensively is in C. first appear in all embryos derived from a heterozygous ogre fe- elegans during the first cleavages. While studies in cultured cells male shortly after the onset of epiboly (4.3 h), just 1.3 h after the and fixed Drosophila tissue depleted of RacGAP1 or MKLP1 zebrafish mid blastula transition (Kane and Kimmel, 1993; Kane homologs by RNAi or mutation indicate cells do not appear to form et al., 1992). Although embryos recover, cytokinesis is abnormally a cleavage furrow (Adams et al., 1998; D’Avino et al., 2006; Le- delayed relative to karyokinesis. Defects begin to appear in komtsev et al., 2012; Mishima et al., 2002; Somers and Saint, homozygous mutants shortly after the onset of gastrulation (6 h), 2003; Sommi et al., 2010; Zavortink et al., 2005), live imaging of C. now 3 h after the zebrafish mid blastula transition. Initially cyto- elegans eggs carrying equivalent mutations show that the first kinesis progresses nearly as far as abscission, before the inter- cleavage furrow forms correctly and ingresses extensively, but cellular bridge between dividing cells regresses and the two then regresses before a stable midbody is established (Canman daughter cells fuse back together, similar to studies in C. elegans. et al., 2008; Davies et al., 2014; Jantsch-Plunger et al., 2000; Eventually, however, mutant cells do not complete cleavage fur- Powers et al., 1998; Raich et al., 1998). Our live imaging studies of row ingression before fusing back together, and finally no

Please cite this article as: Warga, R.M., et al., Progressive loss of RacGAP1/ogre activity has sequential effects on cytokinesis and zebrafish development. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.06.021i R.M. Warga et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 13 cytokinesis occurs. Hence it seems having four or eight nuclei, within perhaps a Centralspindlin is only functional as a complex, not as its in- constrained geometry, compromises the cell and triggers apopto- dividual components (Mishima et al., 2002; Pavicic-Kaltenbrunner sis. Perhaps because mispairing of centrosomes is now more likely et al., 2007), and it is essential for centralspindlin to oligomerize to occur leading to defects in cell polarity or a multipolar spindle and form higher order clusters before becoming active (Basant that does not satisfy the spindle assembly checkpoint (reviewed et al., 2015; Hutterer et al., 2009). Hence, as maternal wild-type in: Bornens (2008), Chavali et al. (2014)) either of which can RacGAP1 is eliminated over time and not replaced by zygotic wild- trigger apoptosis and also contribute to a longer cell cycle type product, the effective concentration of centralspindlin falls (McCaffrey and Macara, 2011; Rieder and Maiato, 2004). offering a unique perspective to study which aspects of cytokinesis Another possible reason cells with four or eight nuclei might are most sensitive to centralspindlin levels. We know of no other die is that after cells have two nuclei DNA damage due to re-re- study that has looked at the result of centralspindlin depletion plication of previously replicated DNA or premature chromosomal over time and our studies indicate that the end of cytokinesis ra- condensation is likelier to occur. This is a common occurrence and ther than the beginning of cytokinesis is exquisitely sensitive to triggers apoptosis during nuclear transfer if the donor nucleus and the amount of centralspindlin available. the recipient cytoplasm are not in phase with each other (reviewed in: Campbell et al. (1996), Gurdon et al. (2003), Shufaro 4.3. Apoptosis in ogre mutants and Reubinoff (2011)). Notably, we see sister nuclei in different phases of the cell cycle using the dual FUCCI cell cycle indicator Loss of function mutations in the homologs of racgap1 or mklp1 (Fig. 5K) or in cdc20/racgap1 mutant cells (Fig. 6E). Therefore, in in Drosophila and C. elegans are embryonic lethal when homo- some mutant cells, each nucleus is progressing through the cell zygous (Adams et al., 1998; Jantsch-Plunger et al., 2000; Powers cycle and its individual checkpoints at different rates. However, et al., 1998; Somers and Saint, 2003; Unhavaithaya et al., 2013; DNA damage is the least likely explanation for ogre mutant cells Zavortink et al., 2005). However, while the cytokinesis defect has dying: homozygous p53 mutants, that do not exhibit apoptosis been much studied in these mutants the cause of lethality has upon DNA damage (Berghmans et al., 2005), do so when injected been largely ignored perhaps because perturbations in cytokinesis with the racgap1 morpholino (R.M.W. unpublished). are apparent well before cells start to die. In contrast, the mutation in the zebrafish homolog of racgap1 was identified initially be- 4.4. RacGAP1 and cellular development cause of massive cell death before it was determined there were earlier defects in cytokinesis (Kane et al., 1996b). Here we show While critical for cytokinesis, little is known of the role of that cell death is due to apoptosis and this is a direct consequence RacGAP1 in other cellular processes. The neuronal defects we of defective cytokinesis for RacGAP1 depleted cells do not die if observe in ogre are especially interesting because they suggest that cytokinesis is prevented using mutants of the cell cycle. While RacGAP1 is needed in various aspects of neuronal development. apoptosis is also observed in the brain of mice and rats carrying a We know from studies in Xenopus when cell division is chemically mutation in citron kinase (Di Cunto et al., 2000; Mitchell et al., blocked early in development that patterning of the nervous sys- 2001; Sarkisian et al., 2002), a contractile ring component needed tem is surprisingly normal and cells even grow axons properly, for successful cytokinesis (Bassi et al., 2013; Gruneberg et al., which elicit typical motor reflexes (Harris and Hartenstein, 1991; 2006), and in the brain of zebrafish carrying a mutation in aurora B Rollins and Andrews, 1991). Therefore it should perhaps not come kinase (Yabe et al., 2009), a chromosomal passenger protein im- as a shock that neurons (and blood) might develop with multiple portant for the completion of cytokinesis (Basant et al., 2015; Guse nuclei as we show. Clearly some defects in ogre stem from defec- et al., 2005) it has never been demonstrated that apoptosis ensues tive cytokinesis, such as the gradual attrition of neuroblasts and because cytokinesis fails. Our results show it does. stem cells over time, which is why in most cell cycle mutants the Why might cells undergoing defective cytokinesis die? In nor- earliest born neurons (primary neurons) are present and later born mal development it is not uncommon for certain cell types to neurons are absent (Fischer et al., 2007; Johnson et al., 2014; Riley become polyploid either as a result of endoreplication or pro- et al., 2010; Song et al., 2004). grammed cytokinesis failure thereby resulting in tetraploid or In general, GTPase-activating proteins similar to RacGAP1, ne- even much greater cell ploidy (reviewed in: Celton-Morizur et al. gatively regulate Rho-mediated signals by stimulating GTP hy- (2010), Fox and Duronio (2013), Orr-Weaver (2015)). And although drolysis of small G-proteins in the Ras superfamily, specifically: cells often die as a result of chromosome missegregation, an ad hoc RhoA, Rac1 and Cdc42. While the major targets of these Rho- mechanism when this occurs is for regression of the cleavage GTPases are the actomyosin and microtubule cytoskeletons (thus furrow yielding a binucleate tetraploid cell rather than two their importance for cytokinesis) by doing so they mediate a di- mononucleate aneuploid cells (Shi and King, 2005). Thus in prin- verse variety of cellular processes that include not only cell cycle ciple, ogre cells should not die as a result of their ploidy, which in progression, but also cell movement, cell signaling, and cell po- some instances has been shown to be an adaptive strategy to larity (reviewed in: Chircop (2014), Citi et al. (2014), Mack and modulate gene expression (Anatskaya and Vinogradov, 2007; Ra- Georgiou (2014)). Accordingly, deregulation of Rho GTPases are slova et al., 2007). However, except for a syncytium where cells linked to a number of neurodegenerative disorders (reviewed in: fuse together to become multinucleate, most vertebrate cells that Stankiewicz and Linseman (2014)). In particular, loss of Rac1 ac- become polyploid rarely have more than two nuclei (reviewed in: tivity has been shown to be a contributing factor in several forms Celton-Morizur et al. (2010), Fox and Duronio (2013), Orr-Weaver of early onset amyotrophic lateral sclerosis (ALS), a devastating (2015)), whereas in ogre they do. Frequently mutant cells go from neuromuscular disorder characterized by loss of motoneurons. two to four nuclei and, less frequently, from four to eight (Fig. 4C). Because the GAP activity of RacGAP1 is specific for Rac1 and not But many, possibly most, mutant cells enter apoptosis during or RhoA (Bastos et al., 2012; Canman et al., 2008; D’Avino et al., 2004; after that division. For, as we show, the majority of mitotic cells are Toure et al., 1998), it is intriguing that while primary motoneurons binucleate rather than quadnucleate (Fig. 4I) and there is a ten- begin to differentiate in ogre, they later die. Other differentiated dency over time for apoptotic cells to out number quadnucleate cells in the spinal cord of the mutant continue to survive including cells (Fig. 7G). Indeed, conditional deletion of RacGAP1 in the floorplate neurons (Fig. 7E′), which lie directly adjacent to primary mouse germline where mutant cells stop dividing once they reach motoneurons (Lewis and Eisen, 2003). Therefore, lack of RacGAP1 two or four nuclei does not cause apoptosis (Lores et al., 2014). likely explains their specific demise. If so, too much Rac1 activity

Please cite this article as: Warga, R.M., et al., Progressive loss of RacGAP1/ogre activity has sequential effects on cytokinesis and zebrafish development. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.06.021i 14 R.M. Warga et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ may be as great a risk to motoneuron survival as too little sug- Motoneuron fate specification revealed by patterned LIM homeobox gene ex- gesting either is a crucial contributing factor to early onset of ALS. pression in embryonic zebrafish. Development 121, 4117–4125. fi Bai, Y., Xiang, X., Liang, C., Shi, L., 2015. Regulating Rac in the nervous system: Unclear is whether motoneuron survival in zebra sh requires molecular function and disease implication of Rac GEFs and GAPs. Biomed. Res. RacGAP1's association with MKLP1. However in Drosophila, de- Int., 632450. pleting motoneurons of Pavarotti (the fly homolog of MKLP1) by Basant, A., Lekomtsev, S., Tse, Y.C., Zhang, D., Longhini, K.M., Petronczki, M., Glotzer, M., 2015. Aurora B kinase promotes cytokinesis by inducing centralspindlin RNAi once motoneurons have begun to differentiate does not ap- oligomers that associate with the plasma membrane. Dev. Cell 33, 204–215. pear to affect their survival (del Castillo et al., 2015) suggesting Bassi, Z.I., Audusseau, M., Riparbelli, M.G., Callaini, G., D’Avino, P.P., 2013. Citron that in this context RacGAP1 acts independently. kinase controls a molecular network required for midbody formation in cyto- – On the other hand, there is growing evidence that the cen- kinesis. Proc. Natl. Acad. Sci. USA 110, 9782 9787. Bastos, R.N., Penate, X., Bates, M., Hammond, D., Barr, F.A., 2012. CYK4 inhibits Rac1- tralspindlin complex has additional functions in axon outgrowth dependent PAK1 and ARHGEF7 effector pathways during cytokinesis. J. Cell fitting with our observations that ogre axons overgrow and their Biol. 198, 865–880. microtubules are disorganized and tangled (Fig. 8). In flies, when Bennett, C.M., Kanki, J.P., Rhodes, J., Liu, T.X., Paw, B.H., Kieran, M.W., Langenau, D. M., Delahaye-Brown, A., Zon, L.I., Fleming, M.D., Look, A.T., 2001. Myelopoiesis the homolog of RacGAP1 is lost, as it is in the tumbleweed mutant, in the zebrafish, Danio rerio. Blood 98, 643–651. axons overgrow (Goldstein et al., 2005), much like we see in ogre. Berghmans, S., Murphey, R.D., Wienholds, E., Neuberg, D., Kutok, J.L., Fletcher, C.D., Notably cytokinesis is mostly normal in tumbleweed mutants Morris, J.P., Liu, T.X., Schulte-Merker, S., Kanki, J.P., Plasterk, R., Zon, L.I., Look, A. fi fi T., 2005. tp53 mutant zebra sh develop malignant peripheral nerve sheath (Goldstein et al., 2005), therefore longer axons seems speci cto tumors. Proc. Natl. Acad. Sci. USA 102, 407–412. loss of RacGAP50C itself. Likewise, if Pavarotti is depleted by RNAi Bierkamp, C., Campos-Ortega, J.A., 1993. A zebrafish homologue of the Drosophila post mitotically (avoiding possible defects from cytokinesis) axons neurogenic gene Notch and its pattern of transcription during early embryogenesis. Mech. Dev. 43, 87–100. are also longer (del Castillo et al., 2015). Recently it has been Bornens, M., 2008. Organelle positioning and cell polarity. Nat. Rev. Mol. Cell Biol. 9, shown that Drosophila neurons initially extend axons by a me- 874–886. chanism involving sliding of microtubules against one another (Lu Bouldin, C.M., Kimelman, D., 2014. Dual fucci: a new transgenic line for studying fi – et al., 2013) and if either Pavarotti or RacGAP50C are depleted this the cell cycle from embryos to adults. Zebra sh 11, 182 183. Brownlie, A., Hersey, C., Oates, A.C., Paw, B.H., Falick, A.M., Witkowska, H.E., Flint, J., stimulates microtubule sliding resulting in longer processes (del Higgs, D., Jessen, J., Bahary, N., Zhu, H., Lin, S., Zon, L.I., 2003. Characterization of Castillo et al., 2015). Therefore centralspindlin appears to act as a embryonic globin genes of the zebrafish. Dev. Biol. 255, 48–61. negative regulator of microtubule dynamics perhaps as suggested Burkard, M.E., Maciejowski, J., Rodriguez-Bravo, V., Repka, M., Lowery, D.M., Clau- ser, K.R., Zhang, C., Shokat, K.M., Carr, S.A., Yaffe, M.B., Jallepalli, P.V., 2009. Plk1 by del Castillo et al. (2015) by crosslinking the microtubules as self-organization and priming phosphorylation of HsCYK-4 at the spindle occurs during cytokinesis. While microtubule sliding has yet to be midzone regulate the onset of division in human cells. PLoS Biol. 7, e1000111. demonstrated in vertebrates, conserved mechanisms for axon Caillat, C., Perrakis, A., 2012. Cdt1 and geminin in DNA replication initiation. Subcell. Biochem. 62, 71–87. outgrowth likely operate as depletion of MKLP1 by RNAi in cul- Campbell, K.H., Loi, P., Otaegui, P.J., Wilmut, I., 1996. Cell cycle co-ordination in tured rat neurons also causes longer axons with a greater number embryo cloning by nuclear transfer. Rev. Reprod. 1, 40–46. of mobile microtubules (Lin et al., 2012). Our results extend this Canman, J.C., Lewellyn, L., Laband, K., Smerdon, S.J., Desai, A., Bowerman, B., Oe- gema, K., 2008. 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