Sarah Webb

Calcium transients triggered by planar signals induce the expression of ZIC3 gene during neural induction in Xenopus

In intact Xenopus embryos, an increase in intracellular Ca(2+) in the dorsal ectoderm is both necessary and sufficient to commit the ectoderm to a neural fate. However, the relationship between this Ca(2+) increase and the expression of early neural genes is as yet unknown. In intact embryos, studying the interaction between Ca(2+) signaling and gene expression during neural induction is complicated by the fact that the dorsal ectoderm receives both planar and vertical signals from the mesoderm. The experimental system may be simplified by using Keller open-face explants where vertical signals are eliminated, thus allowing the interaction between planar signals, Ca(2+) transients, and neural induction to be explored. We have imaged Ca(2+) dynamics during neural induction in open-face explants by using aequorin. Planar signals generated by the mesoderm induced localized Ca(2+) transients in groups of cells in the ectoderm. These transients resulted from the activation of L-type Ca(2+) channels. The accumulated Ca(2+) pattern correlated with the expression of the early neural precursor gene, Zic3. When the transients were blocked with pharmacological agents, the level of Zic3 expression was dramatically reduced. These data indicate that, in open-face explants, planar signals reproduce Ca(2+) -signaling patterns similar to those observed in the dorsal ectoderm of intact embryos and that the accumulated effect of the localized Ca(2+) transients over time may play a role in controlling the expression pattern of Zic3.

On the mechanism of ooplasmic segregation in single-cell zebrafish embryos

It has been previously shown that localized elevations of free cytosolic calcium are associated with a morphological contraction in the forming blastodisc and animal hemisphere cortex during ooplasmic segregation in zebrafish zygotes. It was subsequently proposed, in a hypothetical model, that these calcium transients might be linked to the contraction of a cortically located actin microfilament network as a potential driving force for segregation. Here, by labeling single‐cell embryos during the major phase of segregation with rhodamine‐ phalloidin, direct evidence is presented to indicate that the surface contraction was generated by an actin‐based cortical network. Furthermore, while zygotes incubated with colchicine underwent normal ooplasmic segregation, those incubated with cytochalasin B did not generate a constriction band or segregate to form a blastodisc. During segregation at the single‐cell stage, ooplasm simultaneously moved in two directions: toward the blastodisc within the so‐called axial streamers, and toward the vegetal pole in the peripheral ooplasm. The velocities of both axial and peripheral streaming movements are reported. By injection of a fluorescein isothiocyanate (FITC)‐labeled 2000 kDa dextran into the peripheral ooplasm it was demonstrated that a portion of it feeds into the bases of the extending streamers, which helps to explain the lack of accumulation of ooplasm at the vegetal pole. These new data were incorporated into the original model to link the bipolar ooplasmic movements with the calcium‐modulated, actin‐mediated contraction of the animal hemisphere cortex as a means of establishing and driving ooplasmic segregation in zebrafish.

Calcium signalling during neural induction in Xenopus laevis embryos

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a ‘by default’ mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca2+ imaging of intact Xenopus embryos reveals patterns of Ca2+ transients which are generated via the activation of dihydropyridine-sensitive Ca2+ channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca2+([Ca2+]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca2+]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca2+]i) and then identified early Ca2+-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the ‘by default’ mechanism, where Ca2+ plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca2+-dependent signalling pathways are inactive.

The Use of Aequorins to Record and Visualize Ca2+ Dynamics: From Subcellular Microdomains to Whole Organisms

In this chapter, we describe the practical aspects of measuring [Ca(2+)] transients that are generated in a particular cytoplasmic domain, or within a specific organelle or its periorganellar environment, using bioluminescent, genetically encoded and targeted Ca(2+) reporters, especially those based on apoaequorin. We also list examples of the organisms, tissues, and cells that have been transfected with apoaequorin or an apoaequorin-BRET complex, as well as of the organelles and subcellular domains that have been specifically targeted with these bioluminescent Ca(2+) reporters. In addition, we summarize the various techniques used to load the apoaequorin cofactor, coelenterazine, and its analogs into cells, tissues, and intact organisms, and we describe recent advances in the detection and imaging technologies that are currently being used to measure and visualize the luminescence generated by the aequorin-Ca(2+) reaction within these various cytoplasmic domains and subcellular compartments.

An increase in intracellular Ca2+ is involved in pronephric tubule differentiation in the amphibian Xenopus laevis.

The pronephros is the first kidney to develop and is the functional embryonic kidney in lower vertebrates. It has previously been shown that pronephric tubules can be induced to form ex vivo in ectodermal tissue by treatment with activin A and retinoic acid. In this study, we investigated the role of Ca(2+) signaling in the formation of the pronephric tubules both in intact Xenopus embryos and ex vivo. In the ex vivo system, retinoic acid but not activin A stimulated the generation of Ca(2+) transients during tubule formation. Furthermore, tubule differentiation could be induced by agents that increase the concentration of intracellular Ca(2+) in activin A-treated ectoderm. In addition, tubule formation was inhibited by loading the ectodermal tissue with the Ca(2+) chelator, BAPTA-AM prior to activin A/retinoic acid treatment. In intact embryos, Ca(2+) transients were also recorded during tubule formation, and photo-activation of the caged Ca(2+) chelator, diazo-2, localized to the pronephric domain, produced embryos with a shortened and widened tubule phenotype. In addition, the location of the Ca(2+) transients observed, correlated with the expression pattern of the specific pronephric tubule gene, XSMP-30. These data indicate that Ca(2+) might be a necessary signal in the process of tubulogenesis both ex vivo and in intact embryos.

Visualization of Ca2+ Signaling During Embryonic Skeletal Muscle Formation in Vertebrates

Dynamic changes in cytosolic and nuclear Ca(2+) concentration are reported to play a critical regulatory role in different aspects of skeletal muscle development and differentiation. Here we review our current knowledge of the spatial dynamics of Ca(2+) signals generated during muscle development in mouse, rat, and Xenopus myocytes in culture, in the exposed myotome of dissected Xenopus embryos, and in intact normally developing zebrafish. It is becoming clear that subcellular domains, either membrane-bound or otherwise, may have their own Ca(2+) signaling signatures. Thus, to understand the roles played by myogenic Ca(2+) signaling, we must consider: (1) the triggers and targets within these signaling domains; (2) interdomain signaling, and (3) how these Ca(2+) signals integrate with other signaling networks involved in myogenesis. Imaging techniques that are currently available to provide direct visualization of these Ca(2+) signals are also described.

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.

The pattern of c-fos activation in the CNS is related to behavior in the mudskipper, Periophthalmus cantonensis

The effects of three types of behavior on c-fos activation in different brain regions of the mudskipper, Periophthalmus cantonensis, were studied by immunocytochemistry. Animals were divided into four groups: The control group did not undergo any specific treatment. The second group consisted of animals that were agitated for an hour with a glass rod at an irregular speed. The third group contained animals showing aggressive behavior during an hour of monitoring, i.e. documented territorial hostility by raising of dorsal fins and pursuit of intruders. In contrast to these three groups, which had last been fed 24h earlier, the fourth group included animals that had been nourished 1h prior to sacrifice. Results showed that, in most brain regions of control animals, there were relatively few c-fos positive cells. After fish had been agitated, however, very prominent c-fos label was seen in the lateral and medial parts of the telencephalon, the thalamus, hypothalamus, pituitary and medulla. In aggressive fish, a significant increase in the number of c-fos positive sites, as compared to control fish, was observed in the diencephalons, pons and medulla, but not in the telencephalon. After feeding, there was a less substantial increase in c-fos protein expression in the diencephalon, but an even more prominent c-fos activation in the pons and medulla. Our present results support the hypothesis that, in fish, the medial telencephalon is involved in avoidance reaction and the lateral telencephalon in spatial memory, whereas rhombencephalic activation may reflect activity of cranial nerve nuclei.

Calcium: Calcium signalling during embryonic development

Consider a hypothetical design specification for an integrated communication-control system within an embryo. It would require short-range (subcellular) and long-range (pan-embryonic) abilities, it would have to be flexible and, at the same time, robust enough to operate in a dynamically changing environment without information being lost or misinterpreted. Although many signalling elements appear, disappear and sometimes reappear during development, it is becoming clear that embryos also depend on a ubiquitous, persistent and highly versatile signalling system that is based around a single messenger, Ca2+.

Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca(2+) signaling during myogenesis in intact zebrafish.

We have recently characterized essential inositol 1,4,5-trisphosphate receptor (IP 3R) and ryanodine receptor (RyR)-mediated Ca(2+) signals generated during the differentiation of slow muscle cells (SMCs) in intact zebrafish embryos. Here, we show that the lysosomal two-pore channel 2 (TPC2) also plays a crucial role in generating, and perhaps triggering, these essential Ca(2+) signals, and thus contributes to the regulation of skeletal muscle myogenesis. We used a transgenic line of zebrafish that expresses the bioluminescent Ca(2+) reporter, aequorin, specifically in skeletal muscle, in conjunction with morpholino (MO)-based and pharmacological inhibition of TPC2, in both intact embryos and isolated SMCs. MO-based knock-down of TPC2 resulted in a dramatic attenuation of the Ca(2+) signals, whereas the introduction of TPCN2-MO and TPCN2 mRNA together partially rescued the Ca(2+) signaling signature. Embryos treated with trans-ned-19 or bafilomycin A1, a specific NAADP receptor inhibitor and vacuolar-type H(+)ATPase inhibitor, respectively, also displayed a similar disruption of SMC Ca(2+) signaling. TPC2 and lysosomes were shown via immunohistochemistry and confocal laser scanning microscopy to be localized in perinuclear and striated cytoplasmic domains of SMCs, coincident with patterns of IP 3R and RyR expression. These data together imply that TPC2-mediated Ca(2+) release from lysosomes acts upstream from RyR- and IP 3R-mediated Ca(2+) release, suggesting that the former might act as a sensitive trigger to initiate the SR-mediated Ca(2+)-induced-Ca(2+)-release essential for SMC myogenesis and function.