Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis - PubMed (original) (raw)

Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis

P Kalitsis et al. Genes Dev. 2000.

Abstract

Bub3 is a conserved component of the mitotic spindle assembly complex. The protein is essential for early development in Bub3 gene-disrupted mice, evident from their failure to survive beyond day 6.5-7.5 postcoitus (pc). Bub3 null embryos appear normal up to day 3.5 pc but accumulate mitotic errors from days 4.5-6.5 pc in the form of micronuclei, chromatin bridging, lagging chromosomes, and irregular nuclear morphology. Null embryos treated with a spindle-depolymerising agent fail to arrest in metaphase and show an increase in mitotic disarray. The results confirm Bub3 as a component of the essential spindle checkpoint pathway that operates during early embryogenesis.

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Figures

Figure 1

Figure 1

Targeted disruption of the mouse Bub3 gene. (A) Gene disruption construct and restriction maps. (i) Mouse Bub3 protein showing positions of four WD40 motifs (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-enzyme maps for (ii) the Bub3 gene covering exons 1 to 8, (iii) the neomycin-resistance gene targeting construct, and (iv) the Bub3 locus following targeted disruption. Black boxes represent exons. The selectable marker cassette contained in the targeting construct consists of a splice acceptor site (SA), a picornaviral internal ribosome-entry site (IRES), a lacZ–neomycin fusion gene (beta-geo) and a SV40 polyadenylation sequence (pA). A 1.2-kb _Xba_I–_Eag_I fragment (designated 5′ probe) spanning exon 1 was used in the Southern-screening strategy and detected a 12-kb wild-type _Pst_I fragment in the untargeted locus or a 9.3-kb _Pst_I fragment in the targeted locus. The positions of the primers for nested PCR genotyping of cultured embryos up to day 3.5 + 4 are shown by the 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 ii), giving a final product of 161 bp for the untargeted allele. Beta-geo primer pairs include GF1–GR1 for first-round synthesis and GF2–GR2 for second-round synthesis (open horizontal arrows in iii), giving a final product of 248 bp for the targeted allele. Crosses denote expected sites of homologous recombination. Abbreviations for restriction enzymes are (E) _Eag_I, (P) _Pst_I, (Sc) _Sac_I, (S) _Sal_I, and (X) _Xba_I. (B) Southern blot analysis of wild-type and gene-targeted ES cell lines. The sizes of wild-type 12-kb and homologous recombinant 9.3-kb bands are shown. (C) Nested PCR analysis of cultured 3.5 + 4 day embryos from heterozygous crosses showing the expected bands for the targeted (248 bp) and untargeted (161 bp) alleles.

Figure 2

Figure 2

Morphology of cultured blastocyst outgrowths from heterozygous crosses. Cultured wild-type, heterozygous and homozygous embryos were photographed using phase microscopy, from day 3.5 + 2 to +4. The tightly packed inner cell mass is found in the center of all the embryos (arrow), surrounded by the larger, flat cells of the trophectoderm layer (arrowhead). Scale bar represents 200 μm.

Figure 3

Figure 3

Nuclear morphology of cultured embryos from heterozygous crosses. (A) DAPI-stained day 3.5 + 1 normal embryo, showing regular interphase nuclei and metaphase chromosomes (open arrow). (B,C) Two different magnifications of a DAPI-stained day 3.5 + 1 affected embryo, showing regular interphase nuclei, metaphase chromosomes (open arrow), and micronuclei (arrowheads). (D) A normal embryo at day 3.5 + 2. (E,F) Two different magnifications of an affected embryo at day 3.5 + 2, showing greatly reduced cell number, irregular nuclei with blebs (diamond arrows) and many micronuclei (some examples shown by arrowheads). (G) A normal day 3.5 + 3 embryo containing over 300 cells, with many metaphases (not shown). (H,I) Two different magnifications of a day 3.5 + 3 affected embryo, showing a dramatic reduction in the number of cells and a worsening mitotic phenotype, exhibited by micronuclei, chromatin bridging (closed arrows) and lagging chromosomes (open arrowhead). (J) Anticentromere CREST immunofluorescence of day 3.5 + 2 affected embryos, showing positively stained micronuclei (arrowheads). Scale bars, (A–B) 50 μm, (D,E,G,H) 100 μm, (C,F,I) 20 μm, and (J) 10 μm.

Figure 4

Figure 4

Nocodazole-treated day 3.5+ 1 embryos from heterozygous crosses. (A) A normal embryo, showing regular-sized nuclei and many arrested metaphases (some examples indicated by arrows). (B) A similarly treated null embryo, showing irregularly sized nuclei, very few metaphases (arrow), and a large number of micronuclei (selected examples shown by arrowheads). Scale bars represent 50 μm.

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