Biphasic assembly of the contractile apparatus during the first two cell division cycles in zebrafish embryos.

The large and optically clear embryos of the zebrafish provide an excellent model system in which to study the dynamic assembly of the essential contractile band components, actin and myosin, via double fluorescent labelling in combination with confocal microscopy. We report the rapid appearance (i.e. within <2 min) of a restricted arc of F-actin patches along the prospective furrow plane in a central, apical region of the blastodisc cortex. These patches then fused with each other end-to-end forming multiple actin cables, which were subsequently bundled together forming an F-actin band. During this initial assembly phase, the F-actin-based structure did not elongate laterally, but was still restricted to an arc extending ~15° either side of the blastodisc apex. This initial assembly phase was then followed by an extension phase, where additional F-actin patches were added to each end of the original arc, thus extending it out to the edges of the blastodisc. The dynamics of phosphorylated myosin light chain 2 (MLC2) recruitment to this F-actin scaffold also reflect the two-phase nature of the contractile apparatus assembly. MLC2 was not associated with the initial F-actin arc, but MLC2 clusters were recruited and assembled into the extending ends of the band. We propose that the MLC2-free central region of the contractile apparatus acts to position and then extend the cleavage furrow in the correct plane, while the actomyosin ends alone generate the force required for furrow ingression. This biphasic assembly strategy may be required to successfully divide the early cells of large embryos.