Figure 6. Min mice have an accumulation of tetraploid cells and exhibit tetraploid cells in the small intestine. To determine the ploidy of individual cells in the small intestine, FISH was performed using a probe against chromosome 8. (A–F) A micrograph of a representative cross section from an intestinal crypt is shown with FISH (green) and DAPI (blue) counterstain (A), FISH and FM4-64 (red) membrane counterstain (B), or a merge of FISH, DAPI, and FM4-64 staining (C). The areas indicated by the boxes in C are magniﬁed 2.5-fold and provide examples of a normal diploid cell (D), a G2 cell with four FISH signals (E), and a tetraploid cell (F). (G and H) Min cells with tetrapolar spindle poles in metaphase were visualized using antibodies to Numa (green). Cell boundaries are demarked by β-catenin staining (red), and chromosomes are stained with DAPI. Bars, 10 μm. Discussion Our work shows that clinically relevant mutations in APC act dominantly to inhibit anaphase spindle anchoring, thus re- sulting in cytokinetic failures in dividing intestinal cells in Min mice. This finding implicates a known tumor-initiating mutation in the generation of tetraploid genomes (Fig. 7, red text), a genetic state linked to cancer progression and ge- nomic instability (Fujiwara et al., 2005). The early appear- ance of tetraploid cells in a mouse model for colorectal cancer argues that these dramatic changes are important for cancer progression. A major question that remains unanswered pertains to the fate of intestinal cells with tetraploid genomes. Although we expect that tetraploid cells are inherently unstable (minimally Table I. Tetraploid cells in small intestines Mouse Min Wild type Mina Wild typea Min Min Wild type aIncludes crypts with >70% probe hybridization. Location Crypt Crypt Crypt Crypt Dysplasia Villus Villus ncells 989 903 350 104 218 153 176 because of tetrapolar spindles), the tetraploid model for genomic instability predicts that these cells will give rise to aneuploid cells at the next stage of tumor progression. Unfortunately, it is not trivial to track the fate of a single tetraploid cell in the gut. However, one testable prediction is that dysplasias (Fig. S3) in Min mice will exhibit changes in chromosome ploidy. We find that in contrast to previously reported ploidies of Min tumors (Haigis et al., 2002), dysplasias in Min mice have a notable rate of chromosome gains (4.0% of cells; Table I and see Discussion). This number is likely an underestimate of aneu- ploidy, as we were not confident assigning FISH signals for chromosome loss events in these regions. This finding supports a model (Fig. 7) whereby APC mutants dominantly induce tetra- ploid cells in the crypt that resolve to populate dysplastic regions, thus contributing to genomic instability. Two FISH signals/probe Three FISH signals/probe (%) Four FISH signals/probe (%) 565 457 233 75 132 103 102 65 (6.6) 13 (1.4) 32 (9.1) 5 (4.8) 7 (3.2) 2 (1.3) 0 (0) 31 (3.1) 1 (0.1) 14 (4.9) 0 (0) 4 (1.8) 3 (2.0) 0 (0) APC MUTANTS LEAD TO TETRAPLOID CELLS IN VIVO • CALDWELL ET AL. 1117