Sarah Elizabeth Webb

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+.

Effect of Basic Fibroblast Growth Factor; An In Vitro Study of Tendon Healing

The effect of basic fibroblast growth factor on the proliferative and chemotactic response of cultured rat patellar tendon fibroblasts was studied in an in vitro wound closure model. In quiescent confluent fibroblast culture, a uniform cell free zone, or wound, was generated mechanically as an in vitro wound. The width of the cell free zone was measured at 0, 6, 12, and 24 hours after the injury, in the presence of 0, 2, 10, or 50 ng/mL of basic fibroblast growth factor. Basic fibroblast growth factor, at a concentration of 10 ng/mL, significantly accelerated wound closure, resulting in almost complete closure by 24 hours after the injury. Basic fibroblast growth factor, at a concentration of 2 ng/mL, significantly enhanced cell proliferation as estimated by 5-Bromo-2′-deoxyuridine incorporation, but increasing the concentration of the growth factor to 50 ng/mL did not show additional improvement. Thus, the enhancement of wound closure by basic fibroblast growth factor may be caused by the cell proliferative response, rather than by chemotaxis.

Calcium signalling during zebrafish embryonic development

Calcium signals appear throughout the first 24 hours of zebrafish development. These begin at egg activation, then continue to be generated throughout the subsequent zygote, cleavage, blastula, gastrula, and segmentation periods. They are thus associated with the major phases of pattern formation: cell proliferation, cell differentiation, axis determination, the generation of primary germ layers, the emergence of rudimentary organ systems, and therefore the establishment of the basic vertebrate body plan. When signals need to be transmitted across significant distances they take the form of waves, either intracellular waves when the cell size is large, or later in development when the cell size is reduced, intercellular waves. We will consider both types of calcium signals and their integration into signalling networks, and discuss their possible functions and developmental significance with regard to pattern formation. BioEssays 22:113-123, 2000.

Neurotransmitters, peptides, and neural cell adhesion molecules in the cortices of normal elderly humans and Alzheimer patients: a comparison.

Immunocytochemical techniques was used to compare the proportion of neurons expressing various neurotransmitters (tyrosine hydroxylase, choline acetyltransferase and gamma-aminobutyric acid), neuropeptides (Leu-enkephalin and substance P) and neural cell adhesion molecules (NCAM) in the hippocampus, frontal (area 10) and occipital (area 17) cortices of neurologically normal elderly humans to that of age-matched Alzheimer disease (AD) patients. There was no difference in the proportion of GABAergic and cholinergic cells between the normal and AD groups in all three brain regions studied. However, the catecholaminergic cells in the frontal cortex of the AD patients revealed a significant decrease. The catecholaminergic cells present in the cortex were both neurons and astrocytes, as revealed by a double immunostaining of tyrosine hydroxylase and glial fibrillary acid protein (GFAP). Furthermore, the difference in the proportion of cells expressing Substance P and Leu-enkephalin was minimal between the two groups studied. Although there was little difference in the levels of NCAM in the occipital cortex and hippocampus of the two groups, there were significantly fewer positive NCAM neurons in the frontal cortex of AD than normal aging individuals.

Imaging intercellular calcium waves during late epiboly in intact zebrafish embryos.

Through the injection of f-aequorin and the use of a photon imaging microscope, we have previously reported that a rhythmic series of intercellular Ca2+ waves circumnavigate zebrafish embryos over a 10 h period during gastrulation and axial segmentation. These waves first appear at about 65% epiboly and continue to arise every 5-10 min up to at least the 16-somite stage. In response to our publication, it was suggested that the waves may be an artefact caused by dechorionation of the embryos and would not be observed during the development of intact embryos (i.e. those with chorions). Here we demonstrate (again initially by aequorin imaging) that the rhythmic intercellular Ca2+ waves that traverse the blastoderm margin can also be observed in embryos that have an intact chorion. In addition, the appearance time, propagation pathway, velocity, duration and Ca2+ rise of the waves, as well as the interwave interval and the timing of wave onset, are approximately the same in both dechorionated embryos and those with an intact chorion. Furthermore, by loading intact embryos with Ca(2+)-green dextran at the single-cell stage and then using scanning confocal microscopy to obtain high-resolution images, we confirm the presence of circumferential Ca2+ waves and show that they pass through a population of deep cells located at the blastoderm margin. The confirmation of these pan-embryonic Ca2+ waves in zebrafish further corroborates our earlier suggestion that such waves might play a fundamental role in normal embryonic patterning during the gastrula period.

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.

A role for microtubules in Plasmodium falciparum merozoite invasion

Colchicine, a drug which poisons the polymerization of microtubules, was assayed for effects on the invasion of Plasmodium falciparum merozoites into red cells in order to investigate if merozoite microtubules have a function in invasion. Culture conditions and concentrations of colchicine were established where the maturation and rupture of schizonts was unaffected by the drug. This was judged first by light microscopy, including morphology and counts of nuclear particle numbers, then by ultrastructural studies which excluded deranged organellogenesis as a cause of merozoite failure, and finally by diachronic cultures in which both recruitment and loss of schizonts could be counted. Specific invasion inhibition was seen when 10 microM-1 mM colchicine was present. Red cells pre-incubated in colchicine and then washed showed no reduction in their extent of invasion, and neither red cell lysis, sphering nor blebbing were apparent. We conclude that intact microtubules are necessary for successful merozoite function.

Ca2+ SIGNALLING AND EARLY EMBRYONIC PATTERNING DURING ZEBRAFISH DEVELOPMENT

1 It has been proposed that Ca2+ signalling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern‐forming events during early vertebrate development. 2 With reference to the embryo of the zebrafish (Danio rerio), herein we review the Ca2+ transients reported from the cleavage to segmentation periods. This time‐window includes most of the major pattern‐forming events of early development, which transform a single‐cell zygote into a complex multicellular embryo with established primary germ layers and body axes. 3 Data are presented to support our proposal that intracellular Ca2+ waves are an essential feature of embryonic cytokinesis and that propagating intercellular Ca2+ waves (both long and short range) may play a crucial role in: (i) the establishment of the embryonic periderm and the coordination of cell movements during epiboly, convergence and extension; (ii) the establishment of the basic embryonic axes and germ layers; and (iii) definition of the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen. 4 The potential downstream targets of these Ca2+ transients are also discussed, as well as how they may integrate with other pattern‐forming signalling pathways known to modulate early developmental events.

Calcium signaling during the early development of medaka and zebrafish

The ex-utero fertilization and development of the optically clear embryos of teleost fish have long been favorites of developmental biologists. They have, therefore, provided considerable insight with regards to our understanding of embryonic pattern formation and the early development of vertebrates. These attributes have also been most helpful in the visualization of Ca(2+) signaling events that have been reported to accompany many of the fundamental steps and processes that constitute early embryonic development. These include egg activation; segregation of the cytoplasm from the yolk; cytokinesis; axis determination; cellular rearrangement and germ layer establishment; as well as the formation of the first tissue domains. The developing eggs and embryos of medaka (Oryzias latipes) and zebrafish (Danio rerio) have for many decades been a favorite choice of investigators attempting to visualize Ca(2+) signaling events. In this short review, we have attempted to catalog and present a comparative study of the developmental Ca(2+) signals recorded in these most amenable of vertebrate models.

Calcium signalling during the cleavage period of zebrafish development

Imaging studies, using both luminescent and fluorescent Ca2+-sensitive reporters, have revealed that during the first few meroblastic cleavages of the large embryos of teleosts, localized elevations of intracellular Ca2+ accompany positioning, propagation, deepening and apposition of the cleavage furrows. Here, we will review the Ca2+ transients reported during the cleavage period in these embryos, with reference mainly to that of the zebrafish (Danio rerio). We will also present the latest findings that support the proposal that Ca2+ transients are an essential feature of embryonic cytokinesis. In addition, the potential upstream triggers and downstream targets of the different cytokinetic Ca2+ transients will be discussed. Finally, we will present a hypothetical model that summarizes what has been suggested to be the various roles of Ca2+ signalling during cytokinesis in teleost embryos.