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Fig. 2. lis-1 is required for pronuclear migration, centrosome separation and bipolar spindle assembly. (A-C) Images from time-lapse DIC microscopy of wild-type (A), lis-1(t1550) (B) and lis-1(RNAi) (C) one-cell-stage embryos (see also corresponding Movies 1-3, in supplementary material, http://jcs.biologists.org/cgi/content/full/117/19/4571/DC1). In this and other figures, anterior is to the left, posterior to the right, bars represent 10 µm, and all panels of a kind are at approximately the same magnification, unless stated otherwise. Time elapsed is shown in minutes and seconds, with 00:00 corresponding to the time of maximal pseudocleavage furrow ingression. Arrowheads indicate the position of centrosomes as inferred primarily from fluorescence imaging of equivalent embryos expressing GFP-TUB. Arrows indicate anterior-most boundary of a region depleted of yolk granules that emanates from the centrosomes and extends towards the anterior following breakdown of the male pronuclear envelope. Note that breakdown of the male pronucleus precedes that of the female pronucleus as is generally the case when the two pronuclei are not juxtaposed (Gönczy et al., 1999a). Note also that furrowing initiates but does not go to completion towards the cell anterior in lis-1(t1550) and lis-1(RNAi) embryos, presumably reflecting a local minimum of microtubule density (Dechant and Glotzer, 2003). (D-G) Wild-type one-cell-stage embryo during metaphase (D) and lis-1(t1550) embryo at equivalent stage (E), as well as wild-type four-cell-stage embryo (F) and lis-1(t1550) embryo at equivalent stage (G) stained with antibodies against ZYG-9 (red) and {alpha}-tubulin (green) and counterstained with Hoechst to view DNA (blue). Yellow spots are indicative of ZYG-9 staining at centrosomes (arrowheads). Arrow indicates anterior-most boundary of microtubules emanating from the centrosomes and abutting chromosomes from the male gamete [see also Movie 16 (supplementary material) for similar feature in dhc-1(RNAi) embryo]. Images are projections of two consecutive 1 µm confocal slices. (H) Mean centrosome-to-centrosome distance, measured in live embryos expressing GFP-TUB, in wild-type (n=7), lis-1(RNAi) (n=15) and dhc-1(RNAi) (n=12) embryos at the time of male pronuclear envelope breakdown. Bars represent s.d.