previous characterization of mitosis in MDCK cell monolayers (Reinsch and Karsenti, 1994). We next determined the orientation of mitotic spindles in wild-type mice. We used β-catenin staining to visualize the plasma membrane of the cells and a combination of DAPI and Numa (to visualize spindle poles in mitotic cells) staining to orient the spindle with respect to other cells in the crypt and to the basement membrane (Fig. 4 A). We measured the degree of spindle rotation with respect to the basement membrane (Fig. 4 B). In wild-type cells, 95% of metaphase and 85% of anaphase spindles were oriented at an angle of <30°. Using this value as a cut-off for proper spindle orientation, we observed a dramatic reduction in properly oriented mitotic spindles in Min mice (only 67% of metaphase cells and 53% of anaphase cells had a parallel orientation; P > 0.001; see multiple Min examples in Fig. 4, C and D). This result is consistent with our in vitro observations that spindles in APC mutant cells fail to properly anchor at the cell cortex (Fig. 3). Although we cannot clearly view microtubules in these preparations, we observed phenotypes consistent with micro- tubule defects. We observed that the ratio of the width to length of aligned metaphase chromosomes was increased in Min compared with wild-type mice (Fig. S4, D and E) and that chromosomes were often misaligned and failed to reach the metaphase plate (Fig. S4 F). These results are consistent with previous observations made in vitro and attributed to defective spindle microtubules (Green and Kaplan, 2003). Together, defects in chromosome con- gression, chromosome alignment, and spindle orientation in Min mice strongly argue that a truncating mutation in APC induces mitotic defects in vivo by a similar inhibition of microtubule dynamics as demonstrated in vitro (Green et al., 2005). The mitotic defects we observed in Min mice take place in otherwise normal cells. To more directly address the state of these cells with respect to tumor progression, we determined the fate of the full-length (i.e., wild type) APC protein and the levels of β-catenin in Min mice intestines. We used a carboxy terminal–specific APC antibody that only recognizes the full- length wild-type APC protein (Fig. S5, A and B; available at http://www.jcb.org/cgi/content/full/jcb.200703186/DC1). In crypts from Min mice that exhibit mitotic abnormalities, we observed a characteristic staining of APC at the apical membrane and a basolateral enrichment of β-catenin, patterns that are indistin- guishable from crypt cells in wild-type mice (Fig. 5, A and B). By way of comparison, we observed frequent dysplasias in 7-wk-old Min mice that are easily distinguishable from the normal organization of the crypts (Fig. 5 C, low magnification image). In contrast to normal crypts, we observed the loss of full-length APC and an increase in the cytoplasmic and nuclear β-catenin signal in every dysplasia analyzed (Fig. 5 D). This analysis shows that cells with a wild-type allele of APC and normal levels of β-catenin exhibit mitotic defects and, therefore, argues that APC mutants compromise mitosis independent of β-catenin deregulation. We further confirmed these conclusions by showing that a single mutant allele of APC does not alter β-catenin levels in vitro (Fig. S5 C) and that expression of stabilized β-catenin in normal 293 cells does not result in cyto- kinetic failures (Fig. S5, D–F). As expected, we also observed higher levels of multinucleate human colon tumor cells with APC mutations (SW480) but not in colon tumor cell lines with a stabilizing β-catenin mutation (HCT116; Fig. S1 A). We con- clude that loss of wild-type APC protein is associated with the earliest stages of dysplasia in Min mice and, importantly, that mitotic defects precede the loss of the second allele of APC, β-catenin stabilization, and dysplastic growth. Tetraploid cells in Min mouse intestines Our in vitro data establish a link between defects in spindle orien- tation and failure to induce a cytokinetic furrow. Intriguingly, our observations in Min mice suggest that dividing crypt cells have similar spindle orientation defects, raising the possibility that his- tologically normal crypt cells in Min mice are prone to cyto- kinetic failures. We reasoned that these cytokinetic failures would give rise to tetraploid cells but that these events would be rare. To identify such rare events, we used FISH to determine the copy number of three chromosomes using probes to specific chromo- somal regions (APC probe chromosome 8, p53 probe chromo- some 11, and tex11 probe X chromosome; see Materials and methods FISH section). We used these probes individually and in pair-wise combinations on intestinal tissues from wild-type and Min mice to identify cells with four FISH signals/nucleus. To ensure accuracy in identifying tetraploid cells, we used several methods. To distinguish between neighboring nuclei, we counterstained samples with DAPI and the membrane dye FM4-64. A representative example of a cross section through a Min mouse intestinal crypt is shown in Fig. 6. The FM4-64 dye was used to make the cell boundaries more apparent in the pre- sentation of optical projections. To unambiguously assign FISH signals to individual nuclei, we analyzed stacks of z-sections that included both FISH signal and the DAPI counterstain. Using these approaches, we could easily identify diploid cells and G2 diploid cells by the close proximity of sister chromatid signals (<1 μm; Fig. 6, D and E). Cells with four well-separated FISH signals were interpreted as being tetraploid, although we cannot rule out a near tetraploid genotype (Fig. 6, C and F). We applied stringent criteria for assigning a tetraploid designation, and, in some cases, we ignored potential tetraploid cells if it was not possible to assign their position in a single nucleus; this may have the effect of underestimating the total number of tetraploid cells. Despite this conservative approach, when a large number of crypts was analyzed, we observed a striking number of cells with four distinct FISH probe signals in Min mice (31 cells or 3.1%; Table I; see Discussion). In contrast, there only was one case observed in wild-type mice (0.1%; Table I). Similar trends were observed with all of the FISH probes regardless of whether The dark blue line indicates the position of the basement membrane, and the dashed green line shows the orientation of the spindle. (C) Small intestine crypts stained as in A from wild-type and Min mice (two examples) showing parallel and misoriented mitotic spindles, respectively. (D) Orientation of the mitotic spindle with respect to the basement membrane was determined for >100 mitoses from wild-type and Min mice. The rotational angles were sepa- rated into 5° increments, and the frequency of spindles in each category was plotted. Bars, 10 μm. APC MUTANTS LEAD TO TETRAPLOID CELLS IN VIVO • CALDWELL ET AL. 1115