Papers by Rachel Ashworth
Genome Biology and Evolution
Respiratory electron transport in mitochondria is coupled to ATP synthesis while generating mutag... more Respiratory electron transport in mitochondria is coupled to ATP synthesis while generating mutagenic oxygen free radicals. Mitochondrial DNA mutation then accumulates with age, and may set a limit to the lifespan of individual, multicellular organisms. Why is this mutation not inherited? Here we demonstrate that female gametes-oocytes-have unusually small and simple mitochondria that are suppressed for DNA transcription, electron transport, and free radical production. By contrast, male gametessperm-and somatic cells of both sexes transcribe mitochondrial genes for respiratory electron carriers and produce oxygen free radicals. This germ-line division between mitochondria of sperm and egg is observed in both the vinegar fruitfly and the zebrafishspecies spanning a major evolutionary divide within the animal kingdom. We interpret these findings as an evidence that oocyte mitochondria serve primarily as genetic templates, giving rise, irreversibly and in each new generation, to the familiar energy-transducing mitochondria of somatic cells and male gametes. Suppressed mitochondrial metabolism in the female germ line may therefore constitute a mechanism for increasing the fidelity of mitochondrial DNA inheritance.
Abstract Background Calcium signals ([Ca 2+] i) direct many aspects of embryo development but the... more 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 regul... more 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.
The Journal of physiology, Jan 1, 1999
Briefings in functional genomics & …, Jan 1, 2005
Calcium signals are associated with many of the events common to animal development. Understandin... more Calcium signals are associated with many of the events common to animal development. Understanding the role of these calcium signals requires the ability to visualise and manipulate calcium levels in the developing embryo. Recent work has led to the development of sensitive protein-based probes that can be used to generate transgenic animals for the analysis of calcium signalling in vivo. This paper focuses on the use of genetically encoded calcium probes to follow calcium signals in zebrafish. It reviews progress and speculates on the potential for use in the future.
Integrative Aspects of …, Jan 1, 1998
Journal of Biological …, Jan 1, 2007
IL-15 is a member of the common gamma-chain family of cytokines that possess a heterogeneous repe... more 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.
Journal of cell …, Jan 1, 2005
Developmental Brain Research, Jan 1, 2001
Numerous studies, performed mainly on dissociated cells, have shown that calcium signals have a r... more 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.
Developmental brain research, Jan 1, 2002
Calcium signals play an important role in a variety of processes necessary for neuronal developme... more Calcium signals play an important role in a variety of processes necessary for neuronal development. Whilst the characteristics and function of calcium signals have been comprehensively examined in vitro, the significance of these signals during development in an intact embryo remains unclear. In this study, we have examined the spatial and temporal patterns of intracellular calcium signals in precursor cells (cells without processes) within the spinal cord of the intact zebrafish embryo aged between 17 and 27 h. In total, approximately one-third of cells displayed spontaneous intracellular calcium transients. The calcium transients had an average peak amplitude of 33.3 (62.8%) above baseline, a duration of 52.2 (66.3 s) and occurred with an average frequency of 4.6 (60.4 per hour). Calcium transients were observed in precursor cells located throughout the spinal cord, with the highest percentage of active cells (35.168%) occurring at a developmental time of 21-22 h. Furthermore these intracellular calcium signals were observed in the presence of tricaine, indicating that they are not generated via sodium-dependent action potentials. In precursor cells loaded with the calcium buffer BAPTA both the frequency and the amplitude of the calcium transients was significantly reduced. The intracellular calcium transients may represent a common activity-independent calcium-mediated mechanism that contributes to the regulation of neuronal development in the spinal cord of the zebrafish embryo during the segmentation and early pharyngula period.
Pflügers Archiv European Journal …, Jan 1, 1998
The user has requested enhancement of the downloaded file. All in-text references underlined in b... more The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately.
Cell calcium, Jan 1, 2004
Calcium ions are known to act as important cellular signals during nervous system development. In... more Calcium ions are known to act as important cellular signals during nervous system development. In vitro studies have provided significant information on the role of calcium signals during neuronal development; however, the function of this messenger in nervous system maturation in vivo remains to be established. The zebrafish has emerged as a valuable model for the study of vertebrate embryogenesis. Fertilisation is external and the rapid growth of the transparent embryo, including development of internal organs, can be observed easily making it well suited for imaging studies. The developing nervous system is relatively simple and has been well characterised, allowing individual neurons to be identified. Using the zebrafish model, both intracellular and intercellular calcium signals throughout embryonic development have been characterised. This review summarises technical approaches to measure calcium signals in developing embryonic and larval zebrafish, and includes recent developments that will facilitate the study of calcium signalling in vivo. The application of calcium imaging techniques to investigate the action of this messenger during embryogenesis in intact zebrafish is illustrated by discussion of their contribution to our understanding of neuronal development in vivo.
…, Jan 1, 2008
The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood.... more The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the sloth u45 mutant in which the initial steps in sarcomere assembly take place, but thick filaments are absent and filamentous I-Z-I brushes fail to align or adopt correct spacing. The mutation only affects skeletal muscle and mutant embryos show no other obvious phenotypes. Surprisingly we find that the phenotype is due to mutation in one copy of a tandemly duplicated hsp90a gene. The mutation disrupts the chaperoning function of Hsp90a through interference with ATPase activity. Despite being located only 2kb away from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to down-regulation of genes encoding sarcomeric proteins and upregulation of hsp90a and several other genes encoding proteins that may act with Hsp90a during sarcomere assembly. Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly.
Thyroid, Jan 1, 1998
Primary cultures of rat pituitary cells were stained with an antibody to the native thyrotropin-r... more 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.
Biochemical and …, Jan 1, 1999
Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear phy... more 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.
Trends in endocrinology and …, Jan 1, 1996
The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates... more The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates at low doses. The mechanisms underlying the complex [Ca2+]i response to TRH are discussed. © 1996, Elsevier Science Inc. (Trends
Endocrinology, Jan 1, 1996
Calcium responses to TRH were recorded for individual cells cultured from rat anterior pituitary ... more 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.
Proceedings of the …, Jan 1, 1995
Endocytosis and recycling of both thyrotropin-releasing hormone (TRH) and its G-protein-coupled r... more Endocytosis and recycling of both thyrotropin-releasing hormone (TRH) and its G-protein-coupled receptor were visualized by conventional and confocal fluorescence microscopy in pituitary cells using a rhodaminelabeled TRH analog (Rhod-TRH) and indirect immunofluorescent staining of cells stably transfected with an epitopetagged TRH receptor (TRHR). The epitope-tagged TRHR was confined to the cell surface prior to agonist treatment. Both Rhod-TRH and TRHR were also localized on the plasma membrane after agonist binding at 0°C. Ligand binding at 37C resulted in rapid endocytosis, and both Rhod-TRH and the epitope-tagged TRHR appeared in cytoplasmic vesicles within 5 min. Fluorescently labeled TRH and transferrin colocalized in the same endocytotic vesicles, and internalization of Rhod-TRH and TRHR was inhibited by hypertonic medium, suggesting that endocytosis occurred by a clathrindependent mechanism. Internalized TRHRs returned to the membrane within 20 min after removal of TRH, and cycloheximide did not block receptor recycling. A mutant TRHR truncated at Cys335 signaled but did not internalize Rhod-TRH, confirming the importance of the carboxyl terminus of the TRfIR in receptor-mediated endocytosis. Thus, the TRH-TRHR complex is endocytosed via clathrin-coated vesicles and the receptor is recycled to the plasma membrane.
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Papers by Rachel Ashworth