Ching Man Chan

Multiple roles of the furrow deepening Ca2+ transient during cytokinesis in zebrafish embryos.

The generation of a required series of localized Ca(2+) transients during cytokinesis in zebrafish embryos suggests that Ca(2+) plays a necessary role in regulating this process. Here, we report that cortical actin remodeling, characterized by the reorganization of the contractile band and the formation during furrow deepening of pericleavage F-actin enrichments (PAEs), requires a localized increase in intracellular Ca(2+), which is released from IP(3)-sensitive stores. We demonstrate that VAMP-2 vesicle fusion at the deepening furrow also requires Ca(2+) released via IP(3) receptors, as well as the presence of PAEs and the action of calpains. Finally, by expressing a dominant-negative form of the kinesin-like protein, kif23, we demonstrate that its recruitment to the furrow region is required for VAMP-2 vesicle transport; and via FRAP analysis, that kif23 localization is also Ca(2+)-dependent. Collectively, our data demonstrate that a localized increase in intracellular Ca(2+) is involved in regulating several key events during furrow deepening and subsequent apposition.

Necessary role for intracellular Ca2+ transients in initiating the apical‐basolateral thinning of enveloping layer cells during the early blastula period of zebrafish development

During the early blastula period of zebrafish embryos, the outermost blastomeres begin to undergo a significant thinning in the apical/basolateral dimension to form the first distinct cellular domain of the embryo, the enveloping layer (EVL). During this shape transformation, only the EVL‐precursor cells generate a coincidental series of highly restricted Ca2+ transients. To investigate the role of these localized Ca2+ transients in this shape‐change process, embryos were treated with a Ca2+ chelator (5,5′‐difluoro BAPTA AM; DFB), or the Ca2+ ionophore (A23187), to downregulate and upregulate the transients, respectively, while the shape‐change of the forming EVL cells was measured. DFB was shown to significantly slow, and A23187 to significantly facilitate the shape change of the forming EVL cells. In addition, to investigate the possible involvement of the phosphoinositide and Wnt/Ca2+ signaling pathways in the Ca2+ transient generation and/or shape‐change processes, embryos were treated with antagonists (thapsigargin, 2‐APB and U73122) or an agonist (Wnt‐5A) of these pathways. Wnt‐5A upregulated the EVL‐restricted Ca2+ transients and facilitated the change in shape of the EVL cells, while 2‐APB downregulated the Ca2+ transients and significantly slowed the cell shape‐change process. Furthermore, thapsigargin and U73122 also both inhibited the EVL cell shape‐change. We hypothesize, therefore, that the highly localized and coincidental Ca2+ transients play a necessary role in initiating the shape‐change of the EVL cells.

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.

Inhibition of SOCE disrupts cytokinesis in zebrafish embryos via inhibition of cleavage furrow deepening.

During the first few cell division cycles in zebrafish, distinct Ca(2+) transients are localized to the early embryonic cleavage furrows, where they accompany (and are required for) furrow positioning, propagation, deepening and apposition. It has previously been shown that the endoplasmic reticulum (ER) acts as the primary store for generating these Ca(2+) transients via release through inositol 1,4,5-trisphosphate receptors (IP 3Rs). We hypothesised that maintaining the elevated levels of intracellular Ca(2+) required for deepening and apposition of the cleavage furrows in these large eggs might result in the depletion of the available ER Ca(2+) store, thus the role of store-operated Ca(2+) entry (SOCE) was examined. Newly fertilized, dechorionated embryos were incubated with various SOCE inhibitors, starting just prior to the onset of the first cell division cycle. The effect of these inhibitors on mitosis, furrow positioning, propagation, deepening and apposition, and the generation of the cytokinetic Ca(2+) transients was determined. Treatment with 2-APB or SKF 96365 had no major effect on mitosis, furrow positioning or propagation, but inhibited furrow deepening resulting in regression of the cleavage furrow. Both of these inhibitors also blocked the furrowing Ca(2+) transient, with SKF 96365 having a more profound inhibitory effect than 2-APB. In zebrafish, SOCE does not appear to be required for mitosis or the early stages of cytokinesis during the early embryonic cell division cycles, but it does appear to be essential for maintaining the elevated levels of [Ca(2+)]i for the extended periods that are required during furrow deepening and daughter cell apposition.

Application of complementary luminescent and fluorescent imaging techniques to visualize nuclear and cytoplasmic Ca2+ signalling during the in vivo differentiation of slow muscle cells in zebrafish embryos under normal and dystrophic conditions

1. Evidence is accumulating for a role for Ca2+ signalling in the differentiation and development of embryonic skeletal muscle.

SOCE proteins, STIM1 and Orai1, are localized to the cleavage furrow during cytokinesis of the first and second cell division cycles in zebrafish embryos.

In zebrafish embryos, distinct Ca2+ transients are localized to the early cleavage furrows during the first few cell division cycles. These transients are generated mainly by release via IP3Rs in the endoplasmic reticulum, and they are necessary for furrow positioning, propagation, deepening and apposition. We previously showed, via the use of inhibitors, that store-operated Ca2+ entry (SOCE) also appears to be essential for maintaining the IP3R-mediated elevated levels of [Ca2+]i for the extended periods required for the completion of successful furrow deepening and daughter cell apposition in these large embryonic cells. Here, newly fertilized, dechorionated embryos were fixed at various times during the first and second cell division cycles and immunolabelled with antibodies to STIM1 and/or Orai1 (key components of SOCE). We show that both of these proteins have a dynamic pattern of localization during cytokinesis of the first two cell division cycles. These new data help to support our previous inhibitor results, and provide additional evidence that SOCE contributes to the maintenance of the sustained elevated Ca2+ that is required for the successful completion of cytokinesis in the large cells of embryonic zebrafish.

Introduction of Aequorin into Zebrafish Embryos for Recording Ca2+ Signaling during the First 48 h of Development

Ca(2+) signals, whether transient pulses, propagating waves, or long-duration, steady gradients, are generally considered to play an important role in the pattern-forming events that occur during vertebrate development. One vertebrate that has long been a favorite of embryologists because of its ex utero development and the optical clarity of its embryos is the zebrafish, Danio rerio. Using the bioluminescent Ca(2+) reporter aequorin, distinct Ca(2+) signals have been reported for at least the first 48 h of zebrafish development, with signals becoming progressively more complex as the embryo develops. Here we provide a general introduction to aequorin and its use in monitoring Ca(2+) signals and discuss methods for introducing aequorin into zebrafish embryos.

Identification of Ca 2+ signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells (NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor (IP3R) type-1 and ryanodine receptor (RyR) type-2 are differentially expressed in cells with neuron- or radial glial-like properties. Furthermore, ATP but not caffeine (IP3R and RyR agonists, respectively), induced the generation of Ca2+ transients in cells exhibiting neuron- or glial-like morphology. These results indicate the differential expression of components of the Ca2+-signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca2+ signaling as a read-out.

Reconstitution of Holo-Aequorin with Apoaequorin mRNA and Coelenterazine in Zebrafish Embryos

When holo-aequorin is injected into zebrafish embryos at the one-cell stage, it is normally depleted by ~24 h post-fertilization (hpf). In order to acquire Ca(2+) signaling information from embryos older than 24 hpf, we have developed a protocol to express apoaequorin transiently in embryos, after which we reconstitute active holo-aequorin in vivo by introducing the cofactor coelenterazine into the developing embryo. This protocol describes the preparation of apoaequorin mRNA, followed by microinjection into embryos and incubation with coelenterazine to reconstitute holo-aequorin.