Rachel Ashworth

The ATPase-dependent chaperoning activity of Hsp90a regulates thick filament formation and integration during skeletal muscle myofibrillogenesis

The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the zebrafish slothu45 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 2 kb from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to the downregulation 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.

Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo

Nerve activity is known to be an important regulator of muscle phenotype in the adult, but its contribution to muscle development during embryogenesis remains unresolved. We used the zebrafish embryo and in vivo imaging approaches to address the role of activity-generated signals, acetylcholine and intracellular calcium, in vertebrate slow muscle development. We show that acetylcholine drives initial muscle contraction and embryonic movement via release of intracellular calcium from ryanodine receptors. Inhibition of this activity-dependent pathway at the level of the acetylcholine receptor or ryanodine receptor did not disrupt slow fibre number, elongation or migration but affected myofibril organisation. In mutants lacking functional acetylcholine receptors myofibre length increased and sarcomere length decreased significantly. We propose that calcium is acting via the cytoskeleton to regulate myofibril organisation. Within a myofibre, sarcomere length and number are the key parameters regulating force generation; hence our findings imply a critical role for nerve-mediated calcium signals in the formation of physiologically functional muscle units during development.

Heat shock induces rapid resorption of primary cilia.

Summary Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated 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 loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.

Spontaneous activity-independent intracellular calcium signals in the developing spinal cord of the zebrafish embryo

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 (+/-2.8%) above baseline, a duration of 52.2 (+/-6.3 s) and occurred with an average frequency of 4.6 (+/-0.4 per hour). Calcium transients were observed in precursor cells located throughout the spinal cord, with the highest percentage of active cells (35.1+/-8%) 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.

Buffering intracellular calcium disrupts motoneuron development in intact zebrafish embryos.

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.

Novel 3-nitro-1H-1,2,4-triazole-based compounds as potential anti-Chagasic drugs : in vivo studies

BACKGROUND Chagas disease is caused by the parasite Trypanosoma cruzi, is endemic in Latin America and leads to an estimated 14,000 deaths per year and around 100 million people at risk of infection. Drugs currently used in the treatment of Chagas are old, partially effective and have numerous side effects. METHODOLOGY We have previously reported that 3-nitro-1H-1,2,4-triazole-based compounds demonstrate significant and selective activity against T. cruzi amastigotes in infected L6 cells via activation of a type I nitroreductase, specific to trypanosomatids. In the present work we evaluated in vivo 13 of these compounds based on their high in vitro potency against T. cruzi (IC50 < 1 µM) and selectivity (SI: toxicity to L6 cells/toxicity against T. cruzi amastigotes > 200). Representative compounds of different chemical classes were included. A fast luminescence assay with transgenic parasites that express luciferase, and live imaging techniques were used. A total of 11 out of 13 compounds demonstrated significant antichagasic activity when administered intraperitoneally for 5-10 days at relatively small doses. The best in vivo activity was demonstrated by amides and sulfonamide derivatives. ADMET studies were performed for specific compounds. CONCLUSION At least three compounds were identified as effective, non-toxic antichagasic agents suitable for further development.

Molecular and functional characterization of inositol trisphosphate receptors during early zebrafish development

Fluctuations in cytosolic Ca2+ are crucial for a variety of cellular processes including many aspects of development. Mobilization of intracellular Ca2+ stores via the production of inositol trisphosphate (IP3) and the consequent activation of IP3-sensitive Ca2+ channels is a ubiquitous means by which diverse stimuli mediate their cellular effects. Although IP3 receptors have been well studied at fertilization, information regarding their possible involvement during subsequent development is scant. In the present study we examined the role of IP3 receptors in early development of the zebrafish. We report the first molecular analysis of zebrafish IP3 receptors which indicates that, like mammals, the zebrafish genome contains three distinct IP3 receptor genes. mRNA for all isoforms was detectable at differing levels by the 64 cell stage, and IP3-induced Ca2+ transients could be readily generated (by flash photolysis) in a controlled fashion throughout the cleavage period in vivo. Furthermore, we show that early blastula formation was disrupted by pharmacological blockade of IP3 receptors or phospholipase C, by molecular inhibition of the former by injection of IRBIT (IP3 receptor-binding protein released with IP3) and by depletion of thapsigargin-sensitive Ca2+ stores after completion of the second cell cycle. Inhibition of Ca2+ entry or ryanodine receptors, however, had little effect. Our work defines the importance of IP3 receptors during early development of a genetically and optically tractable model vertebrate organism.

Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development.

BackgroundCalcium signals ([Ca2+]i) direct many aspects of embryo development but their regulation is not well characterised. Ryanodine receptors (RyRs) are a family of intracellular Ca2+ release channels that control the flux of Ca2+ 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. Current evidence suggests that RyRs do not have a major role to play prior to organogenesis but regulate tissue differentiation.FindingsThe sequences of the five zebrafish ryr genes were confirmed, their evolutionary relationship established and the primary sequences compared to other vertebrates, including humans. RyRs are differentially expressed in slow (ryr1a), fast (ryr3) and both types (ryr1b) of developing skeletal muscle. There are two ryr2 genes (ryr2a and ryr2b) which are expressed exclusively in developing CNS and cardiac tissue, respectively. In addition, ryr3 and ryr2a mRNA is detectable in the initial stages of development, prior to embryonic axis formation.ConclusionsOur work reveals that zebrafish ryr genes are differentially expressed throughout the developing embryo from cleavage onwards. The data suggests that RyR-regulated Ca2+ signals are associated with several aspects of embryonic development, from organogenesis through to the differentiation of the musculoskeletal, cardiovascular and nervous system. These studies will facilitate further work to explore the developmental function of RyRs in each of these tissue types.

REAL-TIME MEASUREMENTS OF CALCIUM DYNAMICS IN NEURONS DEVELOPING IN SITU WITHIN ZEBRAFISH EMBRYOS

Abstract We have developed a non-invasive technique to measure intracellular calcium ([Ca2+]i) in neurons growing within intact embryos of the zebrafish (Danio rerio). A single blastomere was injected with a calcium-sensitive fluorescent dye (Calcium Green dextran) between the 32- and 128-cell stage and the embryo imaged between 16 h and 20 h postfertilisation using laser scanning confocal microscopy. Labelled nerve cells from embryos preinjected with dye and dissociated at 16 h showed a fluorescence increase (66±22%; n=11) in response to depolarisation with KCl confirming that the dye remained intracellular and was sensitive to calcium. In addition, fluorescence changes in activated muscle cells of intact embryos showed that the dye was capable of responding to [Ca2+]i changes in vivo. Imaging of dye loaded cells over 30-min periods in embryos between 16 and 20 h revealed that the majority of neurons within the brain and spinal cord did not show spontaneous fluorescence changes distinguishable from noise. However, a subset of neurons within the ventral spinal cord exhibited spontaneous, repetitive [Ca2+]i oscillations which may have a functional significance during neuronal development.

Female and male gamete mitochondria are distinct and complementary in transcription, structure, and genome function

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 gametes—sperm—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 zebrafish—species 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.