bubR1-KEN, bub3¹, and fzy-DYY* mutants exhibit severe defects in broken chromatid segregation. (A) Time-lapse images of neuroblasts expressing I-CreI and labeled with H2A.Z::RFP. The top row shows an example of a dividing cell with equal partition of the broken X chromatids (white arrows), which produces euploid daughter cells. The middle and bottom rows show examples of cells with abnormal segregation of broken chromatids, where three X broken chromatids segregate in one daughter cell (white arrows). These divisions will produce two aneusomic daughter cells with, in some cases, visible micronuclei (yellow arrow, bottom). The pink arrowheads indicate the time at which the last acentric fragments move poleward. Time is given in minutes/seconds. Bars, 10 µm. (B) Histogram showing the frequency of neuroblast divisions with abnormal segregation of broken chromatids after I-CreI expression. n = number of cells. A Fisher extract test was used to calculate the p-value. (#) Given that bub3¹ mutant cells exhibit a high frequency of anaphase with whole lagging chromosomes (Basu et al., 1999; Logarinho et al., 2004; Lopes et al., 2005), cells in which no clear lagging broken chromatids could be followed because of extensive chromosome segregation defects were excluded from our analysis. (C) Scatter dot plot showing the time at which the last acentric chromatid starts moving poleward. The black lines correspond to mean ± 95% confidence interval. Time 0:00 corresponds to anaphase onset. n = number of cells. A Mann-Whitney nonparametric test was used for calculating p-values. (D) DAPI staining of X chromosomes from fixed neuroblasts after I-CreI expression. The bottom row illustrates cells exhibiting two distinct X chromosomes with no apparent tether, indicated by an asterisk, or apparent tether, indicated by pink arrows. The middle row represents intertwined X homologues (cyan arrows), and the top row shows an example of cells with one X broken fragment (yellow arrows). The histogram shows the frequency of cells with either two distinct Xs with or without tethers, intertwined X homologues, or at least one X broken. n = number of cells. Bar, 2 µm. bubR1-KEN(2X) corresponds to bubR1¹ null cells expressing two doses of the RFP::BubR1-KEN transgene; fzy³/+ corresponds to fzy³ heterozygote mutants cells; and fzy³/+ Fzy-DYY* and fzy³/+ Fzy-DYY* (2X) correspond to the fzy³ heterozygote carrying one or two doses of the GFP::Fzy-DYY* transgene.