Figure 5. Mitotic defects occur in histologically normal crypts. (A and B) Intestinal crypts from wild-type (A) and Min (B) mice were stained with β-catenin (red), an anti- body to the C-terminal portion of APC (detects wild-type full-length APC but not truncated APC; see Fig. S5, A and B; available at http://www.jcb.org/cgi/content/full/jcb .200703186/DC1), and DAPI (blue). Arrows indicate the positions of mitotic cells. (C) Low magniﬁcation image of dysplastic regions of the intestine stained as in A. (D) High magniﬁcation image of dysplastic regions stained as in A. The arrows highlight normal β-catenin levels in cells with wild-type APC and elevated β-catenin levels in cells that have lost wild-type APC. Bars, 10 μm. they were used individually or in combination, arguing that these cells minimally contain four copies of chromosomes 8, 11, and X (Fig. 6, Table I, and not depicted). These results are consistent with the single mutant allele of APC in Min mice promoting the formation of tetraploid cells. Because the tetraploid cells we observe are isolated events and are predicted to occur only in the epithelial cells of the small intestine, it is not possible to assess their ploidy using more traditional flow cytometry techniques. Therefore, to con- firm a tetraploid genotype, we looked for additional characteris- tics indicative of tetraploid cell formation. Cells harboring a 4N content of DNA are predicted to have larger nuclei than 2N cells. Analysis of DAPI-stained nuclei showed a consistent trend whereby cells with four FISH signals had nearly two times the cross-sectional area as cells believed to be 2N based on their FISH signals (Table II). Second, failures in cytokinesis leave the resulting cell with two centrosomes; subsequent entry into the next cell cycle induces the duplication of centrosomes and the formation of tetrapolar spindles in the next mitosis. We used Numa staining of centrosomes to identify mitotic cells with more than two poles (Fig. 6 G). Importantly, we observed a large number of tetrapolar spindles only in Min mice (8/110 in Min or 0/105 in wild-type mitotic cells), which is consistent with our in vitro observations of multipolar spindles in cells expressing APC1–1,450 (unpublished data). Together, the FISH and cytological features we observed lead us to conclude that tetraploids form in the intestines of Min mice as a result of cytokinetic failures. 1116 JCB • VOLUME 178 • NUMBER 7 • 2007