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Fig. 2. F-actin dynamics at the furrow is affected by inhibition of myosin activity. Control (A,B,E,F,I,J,M,N), blebbistatin-treated (C,D,G,H,K,L,O,P) and ML7-treated (Q,R) embryos labeled with fluorescent phalloidin. B,F,J,N are higher magnification images of the boxed areas of embryos in A,E,I,M, respectively, and D,H,L,P are similar magnifications of C,G,K,O, respectively. During the early stages of furrow initiation, the contractile ring apparatus forms (arrowhead in B) and appears at a later stage as multiple long cables aligned in the direction of the furrow (arrowhead in F). Formation of the contractile ring apparatus appears relatively normal in blebbistatin-treated embryos (arrowheads in D,H,L), although these embryos can have abnormal f-actin cables perpendicular to the furrow (long arrows in D,H). During furrow formation, control embryos show pericleavage f-actin enrichments that develop into lamella-like structures (short arrows in F). Blebbistatin-treated embryos also show pericleavage f-actin enrichments (short arrows in H), although these do not form normal lamella-like structures. During furrow maturation in control embryos, pericleavage f-actin converges to the center of the furrow (arrowhead in J). On the other hand, in blebbistatin-treated embryos pericleavage f-actin does not converge to the furrow and instead there is an expanded f-actin-free domain between the contractile ring and the pericleavage f-actin enrichments (brackets in L). Towards the end of furrow formation, f-actin forms a compact wall at the furrow in control embryos (N). In blebbistatin-treated embryos at the same stage, pericleavage f-actin domains corresponding to the first furrow have disappeared and f-actin at the contractile apparatus appears as short cables arranged in a ladder-like arrangement that is perpendicular to the plane of the furrow (P). (Q,R) Embryos treated with ML7 also show a reduction in the accumulation of f-actin to the furrow, as well as persistent f-actin enrichments in the region flanking the furrow and short f-actin cables in the furrow region. Images are animal views. In panels with lower magnification images, arrowheads and arrows indicate the first and second cleavage furrows, respectively. Bar, 100 µm (A,C,E,G,I,K,M,O,Q); 16 µm (B,D,F,H,J,L,N,P,R).
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-tubulin (red) antibodies. In control embryos, ß-catenin is initially recruited at the first furrow as aligned punctae of aggregates (arrowheads in A,B) and later forms a contiguous line (arrowheads in E,F,I,J). During these stages, a field of ß-catenin-rich cortical aggregates can be observed centered on the prospective second cleavage furrow (bracket in A). These aggregates will also coalesce into a contiguous line as the second furrow develops (arrow in I). In blebbistatin-treated embryos, the enrichment of aggregates centered on the prospective furrow occurs (bracket in C) and some ß-catenin punctae align at the forming furrow (arrowhead in C,D), but there is no further recruitment of ß-catenin at the furrow as a contiguous structure (arrowheads and arrows in G,H,K,L). Instead, ß-catenin cortical aggregates remain dispersed. A ß-catenin-free region often develops flanking the furrow center (brackets in G), which may correspond to the region where internal membrane has undergone localized exocytosis. Microtubule labeling shows the lack of FMA remodeling in myosin-inhibited embryos (shown in detail in Fig. 4). (M,N) Embryos treated with ML7 also exhibit defects ß-catenin recruitment to the furrow and in FMA remodeling. Arrowheads, arrows and asterisks indicate first, second and third cleavage furrows, respectively. Bar, 50 µm (A,C,E,G,I,K,M) and 16 µm (B,D,F,H,J,L,N).
