Imaging [Ca2+]i with Aequorin Using a Photon Imaging Detector

Self Cite

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca 2+ signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFPmRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca 2+ . Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca 2+ signals from ~2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca 2+ signaling during the 24-48 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca 2+ imaging window by an additional 24 hours.

Section: Introductionmentioning

confidence: 99%

“…Aequorin has also been shown not to interfere with the normal development of living embryos (Miller et al, 1994). This allows for long-term Ca 2+ imaging, which is particularly suited for studying embryonic development (Miller et al, 1994;Ashworth and Brennan, 2005).…”

Section: Introductionmentioning

confidence: 99%

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development

Cheung¹,

Webb²,

Meng³

et al. 2006

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“…technique of Ca 2+ imaging system which has a high sensitivity (Miller et al, 1994). This technique allows visualizing calcium movements in the whole embryo (Fig.…”

mentioning

confidence: 99%

The choice between epidermal and neural fate: a matter of calcium.

Moreau

Leclerc²

2004

The development of the vertebrate embryo, which includes the induction and the patterning of the three germ layers, requires signaling among cells. In vertebrates, cells of the embryonic ectoderm have a choice during gastrulation between two fates; they give rise to epidermal progenitors on the ventral side and neural progenitors on the dorsal side. It was first shown by Spemann and Mangold in 1924 that the dorsal mesoderm (also called the SpemannMangold Organizer) has a potent biological capacity to induce nervous system from the adjacent ectoderm. A similar observation was reported in amniotes, indicating that neural induction by the Organizer is a conserved process controlling the initial steps in vertebrates neurogenesis. Molecular studies of the last decade have led to the identification of signaling molecules which participate in these embryonic decisions. Epidermis induction occurs through a signaling cascade involving Bone Morphogenetic Proteins (BMP 2, 4, 7) and receptor-regulated Smad proteins which translocate into the nucleus to form active transcriptional complexes. These molecules mediate BMPs effect to activate epidermal-specific gene expression and to repress neural-specific gene transcription. Neural fate is revealed by factors secreted by the dorsal mesoderm (Noggin, Chordin, Follistatin, …) which act by blocking BMP signaling. Consequently, epidermal fate is an induced fate while neural fate is interpreted as a default state of the ectoderm. In this review, we describe the signaling pathways which act to determine the fate of the ectoderm and discuss an alternative model in which neural fate is not a default state. This new model integrates the activation of a calcium-dependent signaling pathway due to an influx of calcium through L-type calcium channels. In this model, calcium plays a central regulatory role. While calcium is required for neural determination, epidermal determination occurs when calcium-dependent signaling pathways are inactive.

“…In this way, f-aequorin was carried into the forming blastodisc by ooplasmic streaming (Leung et al, 1998). Aequoringenerated luminescence images and bright-field images were collected with our electron multiplying charge coupled device (EMCCD; Andor, Belfast, Northern Ireland, UK) based photon imaging microscope (PIM) system (custom-made by Science Wares, Falmouth, MA, USA; Miller et al, 1994;Webb et al, 2010), using a Zeiss FLUAR 20x/0.75 N.A. objective lens.…”

Section: Aequorin-based Microinjection and Camentioning

confidence: 99%

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

Chan

Chen²,

Hung³

et al. 2015

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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 3 Rs). 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.