Depletion of esco2 results in genomic instability. (A) Stills taken from time-lapse imaging videos demonstrating the variety of genomic instability observed in 24-hpf esco2 mutant embryos. Micronuclei, anaphase bridges and failed cytokinesis were observed, as well as ‘without error’ divisions. Arrowhead points towards micronuclei. Arrow points to an anaphase bridge. Time stamps are in minutes. (B) Average frequency of above division fates in wild-type (11 divisions taken from three embryos) and esco2 mutant (43 divisions from four embryos) embryos based on time-lapse imaging. Error bars show mean±s.d. between embryos. All wild-type cells underwent a normal division; therefore, there is no s.d. or error bar to report. (C) Western blot of γ-H2AX in protein lysates from 24-hpf esco2+/+ (AB), esco2 sib (+/+ and +/m) and esco2m/m embryos. Irradiated (IR) embryos (100Gy at 24hpf and collected at 30hpf) serve as a positive control. Relative intensity was calculated using ImageJ; each sample was normalized to α-tubulin intensity, and then relative expression was calculated against esco2+/+ (relative normalized intensity=1). (D) Quantification of the number of chromatids within metaphase spreads (ne20 spreads per genotype) from pooled (10-12 embryos) esco2+/+ (AB controls) and esco2m/m embryos. (E) Frequency of micronuclei observed in interphase cells of the tail region of embryos injected with H2afva-eGFP; CAAX-mCherry mRNA. Percentage is based on total number of micronuclei observed over the number of nuclei observed in interphase cells (n=3 embryos/genotype, >75 cells per field, mean±s.d., *P<0.05).
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