Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION tubule-depolymerising agents, resulting in a premature Bub3 gene disruption in mice exit into anaphase before the chromosomes have prop- reveals essential mitotic erly aligned (Chen et al. 1996; Li and Benezra 1996; Tay- spindle checkpoint function lor and McKeon 1997; Cahill et al. 1998; Chen et al. 1998; Gorbsky et al. 1998; Waters et al. 1998; Basu et al. during early embryogenesis 1999; Chan et al. 1999). In humans, dominant-negative mutations of the Bub1 gene show a chromosomal insta- Paul Kalitsis, Elizabeth Earle, Kerry J. Fowler, bility phenotype in colorectal cancer cell lines (Cahill et 1 and K.H. Andy Choo al. 1998). These cell lines also fail to arrest at metaphase Murdoch Children’s Research Institute, Royal Children’s when treated with microtubule-depolymerising drugs. Hospital, Flemington Road, Parkville 3052, Melbourne, Australia Bub3 is found in most eukaryotes through evolution (Efimov and Morris 1998; Taylor et al. 1998; Martinez- Bub3 is a conserved component of the mitotic spindle Exposito et al. 1999). It contains four conserved WD40 assembly complex. The protein is essential for early de- repeats that are found in many proteins with diverse velopment in Bub3 gene-disrupted mice, evident from functions thought to be involved in protein–protein in- their failure to survive beyond day 6.5–7.5 postcoitus teractions (Neer et al. 1994). During mitosis, Bub3 ap- (pc). Bub3 null embryos appear normal up to day 3.5 pc pears on kinetochores during prophase, diminishing in but accumulate mitotic errors from days 4.5–6.5 pc in quantity by metaphase. When kinetochores are unat- the form of micronuclei, chromatin bridging, lagging tached to the spindle, or lagging, the amount of kineto- chromosomes, and irregular nuclear morphology. Null chore-associated Bub3 antigen increases (Martinez-Ex- embryos treated with a spindle-depolymerising agent fail posito et al. 1999). In higher eukaryotes, no mutation or to arrest in metaphase and show an increase in mitotic depletion studies on Bub3 have been described. In this disarray. The results confirm Bub3 as a component of the study, we have performed a genetic disruption of the essential spindle checkpoint pathway that operates dur- Bub3 gene in mouse, using gene-targeting techniques. ing early embryogenesis. We describe a lethal phenotype for the gene-disrupted mice and show that Bub3 is an essential component of Received June 14, 2000; revised version accepted July 25, 2000. the mitotic spindle checkpoint pathway that is required for mitotic fidelity. Results The accurate attachment of chromosomes to the mitotic Disruption of the Bub3 gene spindle via the kinetochore is vital for correct segrega- tion of the genetic material into daughter cells. This To disrupt the mouse Bub3 gene, a promoterless target- process is overseen by a feedback-response mechanism, ing construct was used to obtain a high level of homolo- commonly known as the mitotic spindle checkpoint (for gous recombination. The selection cassette, which con- review, see Skibbens and Hieter 1998; Amon 1999). If tained the splice-acceptor/IRES/lacZ-neomycin resis- defects are detected in the spindle, mitosis is halted to tance gene, was used to delete exons 2 and 3 (Fig. 1A). ensure that chromosomes achieve bipolar alignment be- Only the first 65 amino acids of the 326–amino acid fore the cell proceeds through to anaphase. Bub3 protein would be correctly translated following Genetic screens in the budding yeast Saccharomyces gene disruption with the selection cassette. This disrup- cerevisiae have identified a series of genes (BUB1, BUB2, tion would result in the loss of three of the four WD40 BUB3, MAD1, MAD2, MAD3, and MPS1) that fail to repeat domains and the Bub1–interacting kinetochore arrest in response to spindle damage (Hoyt et al. 1991; Li domain and thus was expected to abolish any Bub3 ac- and Murray 1991; Weiss and Winey 1996). In the pres- tivity at the kinetochore (Taylor et al. 1998). When thecon- ence of microtubule-depolymerising drugs, the mutants struct was transfected into two different ES cell lines,129/1 accumulate severe mitotic errors because of their prema- or W9.5, 20 of 107 (19%) neomycin-resistant cell lines were ture exit into anaphase. Higher eukaryotes contain sev- found to have the desired targeting event (Fig. 1B). eral functional orthologs of these genes, including Bub1, Production of gene-disrupted mice Bub3, BubR1/Mad3, Mad1, and Mad2 (Chen et al. 1996; For the generation of chimeric mice, heterozygous cell Li and Benezra 1996; Taylor and McKeon 1997; Basu et lines from the 129/1 and W9.5 embryonic stem (ES) al. 1998; Chen et al. 1998; Taylor et al. 1998; Basu et al. background were injected into C57BL/6 blastocysts. 1999). Mutations and immunodepletion experiments on This resulted in independent germ line–transmitting Bub1, BubR1/Mad3, Mad1, and Mad2 have shown that chimeric mice from the 129/1 and W9.5 substrains car- cells are unable to block at mitosis in response to micro- rying the targeted allele. The chimeric mice were suc- [Key Words: Mitotic checkpoint; kinetochore; chromosome missegrega- cessfully bred to produce heterozygous animals. tion; mouse transgenic] 1Corresponding author. Embryonic lethality of Bub3 gene-disrupted mice E-MAIL [email protected]; FAX 61-3-9348-1391. +/− Article and publication are at www.genesdev.org/cgi/doi/10.1101/ Bub3 mice were healthy, fertile, and showed no ap- gad.827500. parent phenotype due to haplo-insufficiency. PCR geno- GENES & DEVELOPMENT 14:2277–2282 © 2000 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/00 $5.00; www.genesdev.org 2277 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Kalitsis et al. obtained (Fig. 1C). All 7 −/− embryos, but not any of the +/+ and +/− em- bryos, showed a rapidly degenerating phenotype (see below), suggesting a complete correlation between mor- phological deterioration and the −/− genotype. This result, combined with those for the day 8.5 embryos and the live-born pups, provided evidence that Bub3−/− embryos were viable at day 3.5 in utero and persisted in cul- ture up to day 3.5 + 4 but were com- pletely resorbed or proceeded to de- generate beyond experimental detec- Figure 1. Targeted disruption of the mouse Bub3 gene. (A) Gene disruption construct tion by the time they reached day 8.5 and restriction maps. (i) Mouse Bub3 protein showing positions of four WD40 motifs in utero. (WD) and the Bub1-kinetocore-interacting domain (K) (Taylor et al. 1998; Martinez- Exposito et al. 1999). The disruption site is indicated by the vertical arrow. Restriction- By phase-contrast microscopy, no enzyme maps for (ii) the Bub3 gene covering exons 1 to 8, (iii) the neomycin-resistance significant morphological difference gene targeting construct, and (iv) the Bub3 locus following targeted disruption. Black was observed between the different boxes represent exons. The selectable marker cassette contained in the targeting con- genotypes at day 3.5 or 3.5 + 1 (not struct consists of a splice acceptor site (SA), a picornaviral internal ribosome-entry site shown). In both the wild-type and (IRES), a lacZ–neomycin fusion gene (beta-geo) and a SV40 polyadenylation sequence heterozygous embryos, the transition (pA). A 1.2-kb XbaI–EagI fragment (designated 5Ј probe) spanning exon 1 was used in the from day 3.5 + 2 to 3.5 + 4 was char- Southern-screening strategy and detected a 12-kb wild-type PstI fragment in the untar- acterized by an increase in the size of geted locus or a 9.3-kb PstI fragment in the targeted locus. The positions of the primers the inner cell mass and the trophec- for nested PCR genotyping of cultured embryos up to day 3.5 + 4 are shown by the toderm (Fig. 2). This increase was due horizontal arrows in (ii) and (iii). Wild type primer pairs include BI3H-BI3I for the first round and BI3J-BI3K for the second round of amplification (closed horizontal arrows in to the rapidly dividing inner cell mass ii), giving a final product of 161 bp for the untargeted allele. Beta-geo primer pairs include population, whilst the cells of the GF1–GR1 for first-round synthesis and GF2–GR2 for second-round synthesis (open hori- outer trophectoderm simply in- zontal arrows in iii), giving a final product of 248 bp for the targeted allele. Crosses creased in size without undergoing denote expected sites of homologous recombination. Abbreviations for restriction en- many divisions. In contrast, the inner zymes are (E) EagI, (P) PstI, (Sc) SacI, (S) SalI, and (X) XbaI. (B) Southern blot analysis of cell mass of all the Bub3−/− embryos wild-type and gene-targeted ES cell lines. The sizes of wild-type 12-kb and homologous were significantly smaller in size and recombinant 9.3-kb bands are shown. (C) Nested PCR analysis of cultured 3.5 + 4 day were obviously degenerated by day embryos from heterozygous crosses showing the expected bands for the targeted (248 bp) 3.5 + 3. The trophectoderm, however, and untargeted (161 bp) alleles. appeared morphologically indistin- type analysis of 91 live-born mice from Bub3+/− inter- guishable from those of the wild-type and heterozygous crosses showed 32 wild-type and 59 heterozygote ani- littermates throughout the culture period. mals with no homozygous mutants detected. The Severe mitotic phenotype in null embryos observed wild type:heterozygote:mutant homozygote ra- tio of ∼1:2:0, therefore, suggests an embryonic-lethal To further examine the null phenotype, cultured em- phenotype for the null mutant. bryos from heterozygous crosses were fixed and then To further pinpoint the time of embryonic lethality, stained with DAPI.