Figure 3. WAPL-1 is required for polar body extrusion.
(A) Projections of fixed embryos at the 1- or 2-cell stage stained with DAPI, arrowheads point to the position of polar bodies generated during the meiotic divisions. Polar bodies are found near the cortex in WT control, but one (middle panel) or both (right-hand side panel) polar bodies localize away from the cortex in wapl-1 mutant embryos. (B) Quantification of the percentage of embryos with zero, one, or two polar bodies localized at the cortex (36 embryos scored in wapl-1 and 34 in WT). (C) Quantification of polar body behavior in videos from live WT and wapl-1 mutant embryos expressing a histone H2B::mcherry fusion protein. Note that 7 out of 11 wapl-1 mutant embryos displayed defects in polar body extrusion. Examples of videos used for the quantification are shown in Video 1 (WT), Videos 2–3 (wapl-1). (D) Selected frames from the WT embryo shown in Video 1 and from the wapl-1 mutant embryo shown in Videos 2–3. Time is indicated on top-left corner, starting from metaphase I. Abbreviations: PB I (first polar body), PB II (second polar body), OP (oocyte pronucleus), SP (sperm pronocleus), P0 (first mitotic metaphase following fusion of OP and SP), P1 and AB (cells resulting from the first mitotic division). Note that in the WT embryo PB II remains highly condensed and locates close to PB I on the cortex. In the wapl-1 mutant embryo, PB II becomes decondensed and fails to move to the cortex, first remaining close to the OP and then close to the AB cell produced after the first mitotic division. Chromosomes from AB and PB II appear to mix together before the second mitotic division of the embryo. (E) Projections of fixed embryos following the completion of the second meiotic division and labeled with a FISH probe against the 5S rDNA locus on chromosome V and DAPI. Note that in both WT and wapl-1 mutant embryos the oocyte pronucleus (OP) and the second polar body (PB II) contain a single FISH signal, even when PB II is not localized on the cortex and chromatin appears decondensed (wapl-1 example on right-hand side).
Figure 3—figure supplement 1. Example of abnormal chromatin condensation in polar bodies of wapl-1 mutant embryos.
