Rachel Ashworth

Abstract Background Calcium signals ([Ca 2+] i) direct many aspects of embryo development but their regulation is not well characterised. Ryanodine receptors (RyRs) are a family of intracellular Ca 2+ release channels that control the flux of Ca 2+ from internal stores into the cytosol. RyRs are primarily known for their role in excitation-contraction coupling in adult striated muscle and ryr gene mutations are implicated in several human diseases.

Summary Primary cilia are involved in important developmental and disease pathways, such as regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. Here we investigate the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells cilia loss correlated with a reduction in hedgehog signalling.

IL-15 is a member of the common gamma-chain family of cytokines that possess a heterogeneous repertoire of activities on various cells of the immune system. We report here the first functional characterization of a fish IL-15 in rainbow trout. The trout IL-15 gene is 6-kb long and contains six exons and five introns that transcribe into a 1.2-kb mRNA containing seven out-of-frame AUG initiation codons and translate into a 193-aa peptide. Potential sites for transcriptional activators and repressors have been identified in the trout IL-15 gene. Like IL-15 from other species, trout IL-15 is closely linked to an INPP4B gene, but there is also a BCL10 gene located between the IL-15 and INPP4B genes. Three alternative splicing variants of the trout IL-15 gene have also been identified and their expression in vivo was studied. Trout IL-15 expression is present in all the tissues and cell lines studied. Recombinant trout IFN-gamma selectively increased IL-15 expression but had little effect on other cytokines such as IL-1 beta and IL-11. Recombinant trout IL-15 preferentially stimulated splenic leukocytes from healthy fish, where it induced a large increase in IFN-gamma expression, with little, if any, effect on IL-1 beta expression. This effect was quite long-lived, and was still apparent 24 h poststimulation. Although the exact cell types being affected have still to be determined, it is clear that once produced IL-15 will have a profound affect on the ability of the fish immune system to activate antimicrobial defenses and genes induced themselves by IFN-gamma.

Numerous studies, performed mainly on dissociated cells, have shown that calcium signals have a role during different stages of neuronal development. However, the actions of calcium during neuronal development in vivo remain to be established. The present study has investigated the role of intracellular calcium signals during development of motoneurons in the spinal cord of intact zebrafish embryos. Loading blastomeres of early embryos with either the calcium buffer BAPTA or the calcium reporter dye Calcium Green, was shown to disrupt motoneuron development in the spinal cord of embryos at 24 h postfertilisation. Loading the calcium buffer BAPTA, at an intracellular concentration of 1 mM, into the blastomeres of early embryos did not alter the resting levels of intracellular calcium, but significantly dampened transient rises in intracellular calcium in the cells of later stage embryos. Loading cells with 1 mM BAPTA significantly decreased the number of motoneurons present in the spinal cord at 24 h, indicating that calcium signals are important for normal motoneuron differentiation. Furthermore, in those BAPTA-filled cells that did adopt a motoneuron cell fate, axogenesis was found to be inhibited, suggestive of a role for calcium signalling in neurite initiation. This work provides evidence that calcium signals are necessary at several stages of motoneuron development in vivo.

Primary cultures of rat pituitary cells were stained with an antibody to the native thyrotropin-releasing hormone (TRH) receptor and with a bioactive, fluorescent analogue of TRH, Rhod-TRH. Rhod-TRH specifically stained 86% of lactotropes and 21% of nonlactotropes from primary pituitary cell cultures. Lactotropes and thyrotropes accounted for 90% of cells that stained with Rhod-TRH, but there were occasional lactotropes and thyrotropes that did not show detectable staining with antireceptor antibodies or with Rhod-TRH. The intensity of staining was generally higher in the GH3 line of tumor cells than in normal pituicytes, and 100% of the tumor cells stained with Rhod-TRH. To determine whether the TRH receptor undergoes ligand-directed endocytosis in normal cells, TRH receptor immunocytochemistry was performed before and after TRH binding. TRH receptors were localized on the surface of cells prior to TRH exposure, and Rhod-TRH fluorescence was confined to the plasma membrane when TRH binding was performed at 0 degrees C, where endocytosis is blocked. When cells were incubated with TRH at 37 degrees C, receptors were found in intracellular vesicles in both lactotropes and thyrotropes, and Rhod-TRH was rapidly internalized into endosomes at elevated temperatures. Internalization of Rhod-TRH was inhibited by hypertonic sucrose, indicating that it occurs through clathrin-coated pits. These findings show that some of the heterogeneity in the secretory and calcium responses of pituicytes to TRH occurs at the level of the TRH receptor.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.

Calcium responses to TRH were recorded for individual cells cultured from rat anterior pituitary tissue loaded with fura-2, and cell type was subsequently identified by immunocytochemistry. At 100 nM and 1 microM, TRH stimulated a single transient spike of intracellular free calcium ([Ca2+]i) in 95-100% of lactotrophs. At a concentration of 10 nM or less, the proportion of TRH-responsive cells decreased, and the [Ca2+]i responses became more heterogeneous, consisting of a biphasic response in which an initial [Ca2+]i spike was followed by a sustained elevation of [Ca2+]i or [Ca2+]i oscillations. Initiation of TRH-induced oscillations required the release of intracellular Ca2+ from thapsigargin-sensitive stores, whereas maintenance of the oscillations required influx of extracellular Ca2+ through nimodipine-sensitive Ca2+ channels. The amplitude of the initial [Ca2+]i rise increased from 0.1-10 nM TRH and was not significantly reduced by removal of extracellular Ca2+. The duration of the initial [Ca2+]i transient was significantly shorter at 1 microM than at 1 nM TRH. When TRH was added to cells that had been treated with thapsigargin to block the agonist-induced [Ca2+]i increase, TRH often decreased [Ca2+]i, particularly in cells with high [Ca2+]i. These results suggest that TRH and elevated [Ca2+]i act as coactivators of Ca2+ efflux, which helps terminate the agonist-evoked [Ca2+]i transient. In addition, TRH caused increases in [Ca2+]i in individual rat thyrotrophs, and these responses were heterogeneous. TRH stimulated a [Ca2+]i response in a lesser proportion of thyrotrophs from euthyroid compared to hypothyroid male rats. Essentially all TRH-responsive cells stained for either PRL or TSH.