|
Fig. 1 motley is a mutation in birc5b, which causes early cell division defects in zebrafish embryos.
The cleavage furrow (arrow) seen in live wild-type embryos at 40 mpf during the first cell cycle (A) is absent in motley mutants (B). Early embryonic cytokinesis in zebrafish are rapid and overlap with each other with furrow completion of the previous cell cycle occurring concurrently with the furrow initiation of the next. This results in furrows of various stages of development being present at a single time point in the same embryo (e.g. Figure S1E). During the early cell cycles, immunolabeling for α-tubulin reveal abutting microtubule arrays between nuclei in wild-type blastodisc (C, shown for the furrow corresponding to the first cell cycle), which are abnormal in motley (D). DAPI channel in (C,D) reveals nuclear cycle progresses normally in mutants, although separation of daughter nuclei is reduced likely due to spindle defects (scattered white speckles are caused by autofluorescence by yolk granules, which typically can not be fully removed in the mounting preparation). High magnifications shown here corresponding to the second cell cycles in wild-type and motley show the microtubule-free zone at the furrow in wild-type blastodisc (arrowhead in E, low magnification in Figure S1A), which does not exist in motley (F, low magnification in Figure S1C, boxed area rotated 90° in F). Wild-type mitotic spindle aligns the DNA at the center of the spindle (G, I) while spindles in motley mutants are bent with the DNA spread along their length (H, J). RT-PCRs show that wild-type birc5b is ~500 bp, while motley allele from two different homozygous mutant females is larger at ~600 bp (K). Sequence chromatograms of wild-type (L) and motley (M) birc5b alleles show that the motley sequence is unaltered up to position 236 from the start ATG (vertical line, corresponding to base 305 in the shown sequence chromatograms) and thereafter corresponds to intronic sequence. Schematic of the Birc5b BIR domain that is disrupted due to intron insertion in motley (N).