Search

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.06.029 Byline: Catherine Leclerc (a), Sarah E. Webb (b), Andrew L. Miller (b), Marc Moreau (a) Keywords: Pronephros; Tubulogenesis; Ca.sup.2+ signaling; Ca.sup.2+ imaging; Gene expression; Amphibian Abbreviations: ADPKD, autosomal dominant polycystic kidney disease; BAPTA-AM, N,N'-(1,2-ethanediylbis(oxy-2,1-phenylene))bis(N-(carboxymethyl))-Acetomethyl ester; BCIP/NBT, 5-bromo-4chloro-3-indoyl-phosphate/nitro blue tetrazolium; Diazo-2, N-[2-[2-[2-[bis(carboxymethyl)amino]-5-(diazoacetyl)phenoxy]ethoxy]-4-methylphenyl]-N-(carboxymethyl)-acetomethyl ester; DMSO, dimethyl sulfoxide; hpf, hours post fertilization; MOPS, 3-(N-morpholino)-propanesulfonic acid; NBT, 4-nitro blue tetrazolium chloride; PIM, photon imaging microscope; PMT, photomultiplier tube; TRITC, tetramethyl rhodamine iso-thiocyanate; TRP channel, transient receptor potential channel Abstract: 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.sup.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.sup.2+ transients during tubule formation. Furthermore, tubule differentiation could be induced by agents that increase the concentration of intracellular Ca.sup.2+ in activin A-treated ectoderm. In addition, tubule formation was inhibited by loading the ectodermal tissue with the Ca.sup.2+ chelator, BAPTA-AM prior to activin A/retinoic acid treatment. In intact embryos, Ca.sup.2+ transients were also recorded during tubule formation, and photo-activation of the caged Ca.sup.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.sup.2+ transients observed, correlated with the expression pattern of the specific pronephric tubule gene, XSMP-30. These data indicate that Ca.sup.2+ might be a necessary signal in the process of tubulogenesis both ex vivo and in intact embryos. Author Affiliation: (a) Centre de Biologie du Developpement, UMR 5547 and GDR 2688, Universite Paul Sabatier, 118 Route de Narbonne, F-31062 Toulouse, CEDEX 04, France (b) Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, PRC Article History: Received 20 November 2007; Revised 8 May 2008; Accepted 20 June 2008

We have previously shown that an increase in intracellular [Ca.sup.2+] is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this [Ca.sup.2+] increase and the expression of early neural genes has yet to be defined. Using a subtractive eDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of [Ca.sup.2+], we have isolated the arginine N-methyltransferase, xPRMT1b, a [Ca.sup.2+]-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is [Ca.sup.2+]-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal [Ca.sup.2+]. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a [Ca.sup.2+] increase induces neural fate. calcium | neural induction | protein methyltransferase | Xenopus

In intact Xenopus embryos, an increase in intracellular [Ca.sup.2+] in the dorsal ectoderm is both necessary and sufficient to commit the ectoderm to a neural fate. However, the relationship between this [Ca.sup.2+] increase and the expression of early neural genes is as yet unknown. In intact embryos, studying the interaction between [Ca.sup.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.sup.2+] transients, and neural induction to be explored. We have imaged [Ca.sup.2+] dynamics during neural induction in open-face explants by using aequorin. Planar signals generated by the mesoderm induced localized [Ca.sup.2+] transients in groups of cells in the ectoderm. These transients resulted from the activation of L-type [Ca.sup.2+] channels. The accumulated [Ca.sup.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.sup.2+] -signaling patterns similar to those observed in the dorsal ectoderm of intact embryos and that the accumulated effect of the localized [Ca.sup.2+] transients over time may play a role in controlling the expression pattern of Zic3. Keywords: Neural determination; Planar signals; Calcium signalling; Calcium imaging; Aequorin; Zic3

Background: Voltage‐gated calcium (Cav1) channels contribute to T‐lymphocyte activation. Cav1.2 and Cav1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. Methods: We generated mice deleted for Cav1.2 in T cells or Cav1.3 and analyzed TCR‐driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Cav1.2 or Cav1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav1.2 and Cav1.3 in T cells from asthmatic children correlates with Th2‐cytokine expression. Results: We demonstrated non‐redundant and synergistic functions of Cav1.2 and Cav1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR‐driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Cav1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Cav1.2 and Cav1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav1.2 and Cav1.3 must be co‐expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav1.2 and Cav1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2‐cytokine transcription. Conclusions: Thus, Cav1.2 and Cav1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment. [ABSTRACT FROM AUTHOR]

Increases in intracellular Ca2+ levels (Ca2+)i, promoted by protein kinase C, mediates lectin Con A-triggered neural induction in Pleurodeles waltl embryo. L-type Ca2+ channels are involved in the increases of (Ca2+)i. Neural induction is facilitated by Ca2+-induced Ca2+ release mediated by caffeine and ryanodine. Staurosporine and L-type Ca2+ channel antagonists abolish the (Ca2+)i increase promoted by phorbol ester.

G-proteins associate with neurohormone serotonin (5HT) receptors that stimulate the prophase-blocked oocytes of Ruditapes philippinarum. Inositol triphosphate mass variation is the highest at 3 min, activating the 5HT-sensitive Ca2+ transient. A negative retrocontrol loop with kinase C prevents G proteins binding to 5HT receptors and inhibits Ca2+ production in the metaphase-blocked oocytes.

Intracellular Ca2+ is involved in 5-HT transduction Ruditapes, and the absence of extracellular Ca2+ prevents 5-HT's initiation of maturation in Spisula and Barnea. Ca2+ reserves are utilized in Ruditapes to feed a calcium-induced calcium process initiated by Ca2+ inflow, while in Spisula and Barnea, the calcium reserves increase Ca2+ inflow. Intracellular Ca2+ influx also influences release from prophase and metaphase blocks. The opening of voltage gated Ca2+ channels in Ruditapes prevented meiosin reinitiation in the Ruditapes.