Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION A mutation in separase causes of homozygous cds embryos shows that they also exhibit increased apoptosis as early as 24 hpf, as detected by genome instability and TUNEL staining (Fig. 1B). 5-bromo-2-deoxyuridine increased susceptibility to (BrdU) incorporation also indicates that at 28 hpf, cds embryos have markedly decreased embryonic prolifera- epithelial cancer tion (Supplementary Fig. S1). To further characterize the larger foci phenotype and cell cycle defects exhibited by 1,3 1,4 Jennifer L. Shepard, James F. Amatruda, cds mutant embryos, DNA content analysis was per- David Finkelstein,1 James Ziai,1 K. Rose Finley,1 formed on disaggregated 24-hpf embryos. This study Howard M. Stern,1,2 Ken Chiang,1 demonstrated that cds embryos have a population of Candace Hersey,1 Bruce Barut,1 cells with 8N DNA content, indicating the presence of 2 2 polyploid cells (Fig. 1C). Further analysis also demon- Jennifer L. Freeman, Charles Lee, strated the presence of 16N cells (data not shown). These 2 2 Jonathan N. Glickman, Jeffery L. Kutok, data suggest that cds mutants have a fundamental prob- Jon C. Aster,2 and Leonard I. Zon1,5 lem with control of the mitotic checkpoint. To discover the gene responsible for the cds pheno- 1Children’s Hospital, Boston, Massachusetts 02115, USA; 2 type, a positional cloning project was undertaken and Department of Pathology, Brigham and Women’s Hospital, cds was mapped and localized to linkage group 6 in a Boston, Massacusetts 02115, USA 6.5-centimorgan (cM) interval between the microsatel- lite markers z5294 and z265 (Fig. 1D). Scanning of addi- Proper chromosome segregation is essential for mainte- tional microsatellite makers narrowed the interval to 0.5 nance of genomic integrity and instability resulting from cM. A chromosomal walk was initiated from z11919, failure of this process may contribute to cancer. Here, we and led to the identification of a single BAC clone that demonstrate that a mutation in the mitotic regulator contained the cds locus. Analysis of BAC sequence and separase is responsible for the cell cycle defects seen in human/zebrafish syntenic relationships led to the dis- the zebrafish mutant, cease&desist (cds). Analysis of cds covery that the zebrafish ortholog of the vertebrate gene homozygous mutant embryos reveals high levels of poly- separase was present in this critical interval. Sequence ploidy and aneuploidy, spindle defects, and a mitotic exit analysis of the cds mutant locus identified a C-to-A delay. Carcinogenesis studies demonstrated that cds het- transversion introducing a nonsense mutation that is predicted to truncate the zebrafish separase gene after erozygous adults have a shift in tumor spectrum with an amino acid 597, which would cause loss of the catalytic eightfold increase in the percentage of fish bearing epi- domain and loss of half of the ARM repeats at the N thelial tumors, indicating that separase is a tumor sup- terminus (Viadiu et al. 2005). Separase is a cysteine pro- pressor gene in vertebrates. These data strongly support tease that, during mitosis, is responsible for cleaving the a conserved cross-species role for mitotic checkpoint cohesin complex that binds sister chromatids together genes in genetic stability and epithelial carcinogenesis. (Nasmyth 2001). Separase is tightly regulated by an ana- phase-promoting complex target protein, securin, which Supplemental material is available at http://www.genesdev.org. has been shown to act as the oncogene pituitary tumor Received July 18, 2006; revised version accepted November 8, transforming gene (PTTG) in humans (Zou et al. 1999). 2006. Separase is also regulated by autocatalytic cleavage and inhibitory phosphorylation (Stemmann et al. 2001; Wai- zenegger et al. 2002; Zou et al. 2002). Additionally, in Results and Discussion yeast, separase has been shown to act as part of the mi- A previously described genetic screen in zebrafish for totic exit network (MEN) complex, suggesting that sepa- cell proliferation mutants identified cease&desist (cds), rase simultaneously triggers anaphase by cohesin cleav- a homozygous lethal mutant with aberrant staining for age and, independently of its protease function, initiates the phosphorylated form of histone H3 (pH3), a mitotic mitotic exit through activation of Cdc14 (Stegmeier et marker (Shepard et al. 2005). pH3 staining of cds em- al. 2002). Zebrafish separase encodes a 2181-amino-acid bryos 36 h post-fertilization (hpf) shows a slight decrease protein that is 34% identical to human separase, and in the number of pH3-positive cells (Fig. 1A) and reveals 13% identical to Schizosaccharomyces pombe Cut1 and that these pH3-positive foci are also larger than their Saccharomyces cervisiae Esp1 (Supplementary Fig. S2). wild-type sibling counterparts. Further characterization The catalytic residues that are present in all caspase- hemoglobinase fold family members are also conserved (Aravind and Koonin 2002). DNA content analysis of wild-type embryos injected with antisense morpholino- [Keywords: Cancer; chromosome segregation; mitotic checkpoint; ze- brafish] modified oligonucleotides (morpholinos) targeted to the Present addresses: 3Center for Cancer Research, Massachusetts Institute separase exon 7 splice donor shows that ∼30% of the of Technology, Cambridge, MA 02139, USA; 4Departments of Pediatrics cells are polyploid, which is similar to the level seen in and Molecular Biology, University of Texas Southwestern Medical Cen- cds homozygous mutants (Fig. 1E). These data strongly ter, 5323 Harry Hines Blvd., Dallas, TX 75390, USA. 5Corresponding author. suggest it is a loss of function of separase that is respon- E-MAIL [email protected]; FAX (617) 730-0222. sible for the cds phenotype. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1470407. Loss of separase in yeast, human, and mouse cells has GENES & DEVELOPMENT 21:55–59 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org 55 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Shepard et al. extra time to reach the pole (Pidoux et al. 2000). Our studies have demonstrated that such a mi- totic exit defect can be detected. To examine the cell cycle timing of cds cells from S to M phase and determine if a mitotic entry or exit defect existed, we performed a BrdU pulse/chase experi- ment and double stained the embryos with anti- BrdU antibody and pH3 antibody. Examining the timing of when BrdU-positive cells become pH3 positive allows calculation of the length of time between the S phase and mitotic entry and exit in cds as compared with wild types (Fig. 2J). The S-phase cell population in wild-type embryos be- gins to enter G2/M 2 h after BrdU labeling and begins to exit from mitosis at 6 h, with exit com- pleted by 8 h. Cells in cds embryos enter M phase at the same time as wild types, and also begin to exit at 6 h post-incorporation. There are still a significant proportion of cds cells in mitosis at the 8-h time point, indicating slower progression through mitosis, perhaps due to mitotic compli- cations, or a potential mitotic exit defect. Sepa- rase has been shown to act as part of a complex that promotes mitotic exit in yeast (Stegmeier et al. 2002), so this phenotype is consistent with a similar role in vertebrates. However, recent stud- Figure 1. cds has mitotic cells with increased size of pH3 foci and increased ies in mouse embryonic fibroblasts lacking Sepa- apoptosis and polyploidy, and is caused by a mutation in the zebrafish separase rase have not uncovered any mitotic exit defects gene. (A) High-power views of a 36-hpf cds mutant show that pH3-positive foci (Kumada et al. 2006). This discrepancy between in cds are larger in size than in wild-type siblings. (B) TUNEL staining of 24-hpf cds embryos shows increased apoptosis in the head and dorsal part of the tail. (C) zebrafish embryos and mouse embryonic fibro- DNA content analysis of cds homozygous 36-hpf embryos and wild-type siblings blasts (MEFs) may be attributed to studying this showing the presence of polyploid cells in the mutant. (D) Positional cloning of phenomenon in a whole organism versus in a cell the cds gene. The cds locus is depicted by a black bar between the flanking culture environment; the defect may only be vis- markers z11919 and z7248 identified by analysis of ∼3000 meioses from diploid ible in the former. mutants. Below is an enlarged view of the cds locus that depicts the BAC ge- To further characterize the polyploidy seen in nomic clones identified in a chromosomal walk. The number of recombination events identified from the distal side (squares) and proximal side (circles) are cds embryos and to look for further evidence of indicated. (E) Phenocopy of the cds polyploid phenotype by injection of morpho- chromosomal instability as indicated by the pres- linos targeted to the separase exon 14 splice donor. ence of grossly abnormal mitoses, interphase fluorescent in situ hybridization (FISH) analysis was performed using probes specific to two ran- been shown to cause mitotic spindle defects such as domly chosen linkage groups. This analysis shows that multiple spindle poles (Baum et al. 1988; Uzawa et al. cds embryos contain 48% polyploid and 34% aneuploid 1990; Waizenegger et al. 2002; Chestukhin et al. 2003; cells versus 2% and 7%, respectively, in wild types (Fig. Kumada et al. 2006; Wirth et al. 2006). Examination of 3A,B). Further analysis of metaphase spreads demon- the mitotic spindle in 32-hpf cds mutants demonstrated strated the presence of cells in cds mutant embryos with that ∼50% of the visible spindles were abnormal (Fig.