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Andrew L Miller

Publications and source records attributed to Andrew L Miller.

15 recordsLinked to original sources
PubMed2006

Calcium transients and calcium signalling during early neurogenesis in the amphibian embryo Xenopus laevis.

Development of the vertebrate embryonic nervous system is characterized by a cascade of signalling events. In Xenopus, the initial step in this cascade results from signals emanating from the dorsal mesoderm that divert the fate of the ectoderm from an epidermal to a neural lineage. These signals include extracellular antagonists of the bone morphogenetic protein (BMP). Experiments performed with isolated ectoderm suggest that epidermis is induced by BMP, whereas neural fates arise by default following BMP inhibition; however, we show that this mechanism is not sufficient for neural determination. Ca2+ imaging of intact Xenopus embryos reveals patterns of Ca2+ transients in the dorsal ectoderm but not in the ventral ectoderm. These increases in intracellular calcium concentration ([Ca2+](i)), which occur via the activation of dihydropyridine (DHP)-sensitive Ca2+ channels, are necessary and sufficient to orientate the ectodermal cells toward a neural fate. On the one hand, the treatments that antagonize the increase in [Ca2+](i), inhibit neuralization, while on the other hand, an artificial increase in [Ca2+](i), whatever its origin, neuralizes the ectoderm. Using these properties, we have constructed a subtractive cDNA library between untreated ectoderm and caffeine-treated ectoderm. The caffeine stimulates an increase in [Ca2+](i) and thus orientates the cells towards the neural pathway. We have identified early Ca2+ target genes expressed in neural territories. One of these genes, an arginine methyl transferase, controls the expression of the early proneural gene, Zic3. Here, we discuss an alternative model where Ca2+ plays a central regulatory role in early neurogenesis. This model integrates the activation of a Ca2+ -dependent signalling pathway due to an influx of Ca2+ through DHP-Ca2+ channels. While Ca2+ is required for neural determination, epidermal determination occurs when Ca2+ -dependent signalling pathways are inactive.

Animals↗
PubMed2006

Ca2+ signaling and early embryonic patterning during the blastula and gastrula periods of zebrafish and Xenopus development.

It has been proposed that Ca(2+) signaling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern forming events during early vertebrate development [L.F. Jaffe, Organization of early development by calcium patterns, BioEssays 21 (1999) 657-667; M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11-21; S.E. Webb, A.L. Miller, Calcium signalling during embryonic development, Nat. Rev. Mol. Cell Biol. 4 (2003) 539-551]. With reference to the embryos of zebrafish (Danio rerio) and the frog, Xenopus laevis, we review the Ca(2+) signals reported during the Blastula and Gastrula Periods. This developmental window encompasses the major pattern forming events of epiboly, involution, and convergent extension, which result in the establishment of the basic germ layers and body axes [C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253-310]. Data will be presented to support the suggestion that propagating waves (both long and short range) of Ca(2+) release, followed by sequestration, may play a crucial role in: (1) Coordinating cell movements during these pattern forming events and (2) Contributing to the establishment of the basic embryonic axes, as well as (3) Helping to define the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen [E. Gilland, A.L. Miller, E. Karplus, R. Baker, S.E. Webb, Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation, Proc. Natl. Acad. Sci. USA 96 (1999) 157-161; J.B. Wallingford, A.J. Ewald, R.M. Harland, S.E. Fraser, Calcium signaling during convergent extension in Xenopus, Curr. Biol. 11 (2001) 652-661]. The various potential targets of these Ca(2+) transients will also be discussed, as well as how they might integrate with other known pattern forming pathways known to modulate early developmental events (such as the Wnt/Ca(2+)pathway; [T.A. Westfall, B. Hjertos, D.C. Slusarski, Requirement for intracellular calcium modulation in zebrafish dorsal-ventral patterning, Dev. Biol. 259 (2003) 380-391]).

Animals↗
PubMed2006

Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos.

Cytokinesis is the final stage in cell division that serves to partition cytoplasm and daughter nuclei into separate cells. Membrane remodeling at the cleavage plane is a required feature of cytokinesis in many species. In animal cells, however, the precise mechanisms and molecular interactions that mediate this process are not yet fully understood. Using real-time imaging in live, early stage zebrafish embryos, we demonstrate that vesicles labeled with the v-SNARE, VAMP-2, are recruited to the cleavage furrow during deepening in a microtubule-dependent manner. These vesicles then fuse with, and transfer their VAMP-2 fluorescent label to, the plasma membrane during both furrow deepening and subsequent apposition. This observation indicates that new membrane is being inserted during these stages of cytokinesis. Inhibition of SNAP-25 (a cognate t-SNARE of VAMP-2), using a monoclonal antibody, blocked VAMP-2 vesicle fusion and furrow apposition. Transient expression of mutant forms of SNAP-25 also produced defects in furrow apposition. SNAP-25 inhibition by either method, however, did not have any significant effect on furrow deepening. Thus, our data clearly indicate that VAMP-2 and SNAP-25 play an essential role in daughter blastomere apposition, possibly via the delivery of components that promote the cell-to-cell adhesion required for the successful completion of cytokinesis. Our results also support the idea that new membrane addition, which occurs during late stage cytokinesis, is not required for furrow deepening that results from contractile band constriction.

Animals↗
PubMed2006

Requirement for a localized, IP3R-generated Ca2+ transient during the furrow positioning process in zebrafish zygotes.

We report that the first localized Ca(2+) transient visualized in the blastodisc cortex of post-mitotic zebrafish zygotes has unique features. We confirm that this initial 'furrow positioning' Ca(2+) transient precedes the physical appearance of the first cleavage furrow at the blastodisc surface and that it has unique dynamics, which distinguish it from the subsequent furrow propagation transients that develop from it. This initial transient displays a distinct rising phase that peaks prior to the initiation of the two linear, subsurface, self-propagating Ca(2+) waves that constitute the subsequent furrow propagation transient. Through the carefully timed introduction of the Ca(2+) buffer, dibromo-BAPTA, we also demonstrate the absolute requirement of this initial rising phase Ca(2+) transient in positioning the furrow at the blastodisc surface: no rising phase transient, no cleavage furrow. Likewise, the introduction of the inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, 2-aminoethoxydiphenyl borate, eliminates both the rising phase transient and the appearance of the furrow at the cell surface. On the other hand, antagonists of the ryanodine receptor and NAADP-sensitive channels, or simply bathing the zygote in Ca(2+)-free medium, have no effect on the generation of the rising phase positioning transient or the appearance of the furrow at the surface. This suggests that like the subsequent propagation and deepening/zipping Ca(2+) transients, the rising phase furrow positioning transient is also generated specifically by Ca(2+) released via IP3Rs. We propose, however, that despite being generated by a similar Ca(2+) release mechanism, the unique features of this initial transient suggest that it might be a distinct signal with a specific function associated with positioning the cleavage furrow at the blastodisc surface.

Aequorin↗
PubMed2006

Ca2+ signaling during vertebrate somitogenesis.

A variety of Ca2+ signals, in the form of intercellular pulses and waves, have been reported to be associated with the various sequential stages of somitogenesis: from convergent extension and the formation of the paraxial mesoderm; during the patterning of the paraxial mesoderm to establish segmental units; throughout the formation of the morphological boundaries that delineate the segmental units, and finally from within the maturing somites as they undergo subsequent development and differentiation. Due to both the technical challenges presented in imaging intact, developing embryos, and the subtle nature of the Ca2+ transients generated, they have proved to be difficult to visualize. However, a combination of cultured cell preparations and improvements in explant and whole embryo imaging techniques has begun to reveal a new and exciting class of developmental Ca2+ signals. In this chapter, we review the small, but expanding, number of reports in the literature and attempt to identify common characteristics of the somitogenic Ca2+ transients, such as their mode of generation, as well as their spatial and temporal features. This may help to elucidate the significance and function of these intriguing Ca2+ transients and thus integrate them into the complex signaling networks that orchestrate early developmental events.

Animals↗
PubMed2006

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

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::EGFP -mRNA 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 2448 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca(2+) imaging window by an additional 24 hours.

Aequorin↗
PubMed2005

Calcium transients and neural induction in vertebrates.

Evidence indicates that a variety of different types of Ca2+ transients (i.e., standing gradients, pulses and propagating waves) may be occurring both simultaneously and sequentially during neural induction in vertebrate embryos. Transients have been observed in the dorsal marginal zone and in the presumptive neural ectoderm, suggesting that they may be generated within two distinct germ layers at separate embryological locations. It has been proposed that the Ca2+ transients might have multiple roles during the period of neural induction, ranging from: activating the expression of early neural genes; contributing to the inhibition of BMP-4 signalling; generating secretion gradients of morphogens; regulating and co-ordinating convergent extension; and establishing and reinforcing dorsoventral axis specification. Both intra- and extracellular stores (either acting separately or in combination) have been shown to generate the neuralizing Ca2+ transients via well-established release mechanisms, and transients have been shown to propagate between connected cells, suggesting an intercellular signalling dimension. Thus, good evidence is accumulating to suggest that Ca2+ might be a key central regulator in the process of neural induction.

Animals↗
PubMed2004

Organization and function of microfilaments during late epiboly in zebrafish embryos.

We report that, during epiboly in zebrafish, three F-actin--based structures appear only after the blastoderm migrates past the embryonic equator. They are composed of two ring-like F-actin structures that form at the deep cell and enveloping layer margins of the blastoderm and a punctate actin band that develops in the external yolk syncytial layer. Treatment with cytochalasin B or the calcium chelator dibromo-BAPTA results in the disruption of all three of these actin-based structures, leading to the slowing or immediate arrest of epiboly, respectively, followed by a failure of yolk cell occlusion and the eventual lysis of the embryo through the vegetal pole region. We suggest, therefore, that these structures function in the occlusion of the vegetal portion of the yolk cell during the latter stages of epiboly. Possible roles for these new structures, their modulation by Ca2+, as well as the functions of other previously described F-actin--based structures observed throughout epiboly, are discussed.

Actin Cytoskeleton↗
PubMed2004

Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its (Ca2++Mg2+)ATP-ases in the activation of store-operated Ca2+ channels in liver cells.

The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2++Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Cao2+) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Cao2+-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Cao2+-induced [Ca2+]cyt spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Cao2+-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2++Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.

Aequorin↗
PubMed2004

Characterization of mid-spindle microtubules during furrow positioning in early cleavage period zebrafish embryos.

We report evidence to suggest that during the first few meroblastic cell divisions in zebrafish embryos a dynamic population of central-spindle microtubules serve a crucial function in positioning the cleavage furrow at the surface of the blastoderm. Originating from the mid-zone of the mitotic spindle they develop into what we term a mid-spindle 'pre-furrowing microtubule array' that expands upward and outward from the spindle mid-zone towards the blastodisc surface. We suggest that this structure transmits positional information to the blastodisc cortex that results in the correctly positioned assembly of the cytokinetic contractile apparatus. We also propose that the pre-furrowing microtubule array then develops into a furrow-ingression microtubule array that helps direct and assemble the deepening furrow as it cuts its way through the blastodisc. Due to the location of its origin, the pre-furrowing microtubule array serves to successfully separate the daughter nuclei and thus equally divide the blastoderm. Furthermore, co-localization with elements of the cortical endoplasmic reticulum and their inositol 1,4,5-trisphosphate receptors suggests that the pre-furrowing microtubule array may also play a role in organizing localized Ca2+ transients that have been shown to be essential to the furrow positioning, propagation and deepening process during cytokinesis in zebrafish embryos.

Animals↗
PubMed2003

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.

Aequorin↗
PubMed2003

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.

Aequorin↗
PubMed2003

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+.

Animals↗
PubMed2003

Ca2+ released via IP3 receptors is required for furrow deepening during cytokinesis in zebrafish embryos.

We have previously visualized three Ca2+ transients, generated by release from intracellular stores, which are associated with cytokinesis during the early cell division cycles of zebrafish embryos: the furrow positioning, propagation and deepening transients. Here we demonstrate the requirement of the latter for furrow deepening, and identify the Ca2+ release channels responsible for generating the deepening transient. The introduction of the Ca2+ buffer 5,5'-dibromo-BAPTA, at an appropriate time to challenge only the deepening transient, resulted in the dissipation of this transient and an inhibition of furrow deepening. Introduction of antagonists of the inositol 1,4,5-trisphosphate (IP3) receptor (heparin and 2-aminoethoxydiphenylborate; 2-APB) at the appropriate time, blocked the furrow deepening transient and resulted in an inhibition of furrow deepening. In contrast, antagonists of the ryanodine receptor and the NAADP-sensitive channel had no effect on either the furrow deepening transient or on furrow deepening. In addition, microinjection of IP3 led to the release of calcium from IP3-sensitive stores, whereas the introduction of caffeine or cADPR failed to induce any increase in intracellular Ca2+. Our new data thus support the idea that Ca2+ released via IP3 receptors is essential for generating the furrow deepening transient and demonstrate a requirement for a localized cytosolic Ca2+ riseforthe furrow deepening process. We also present data to show that the endoplasmic reticulum and IP3 receptors are localized on either side of the cleavage furrow, thus providing the intracellular Ca2+ store and release mechanism for generating the deepening transient.

Animals↗