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First published online 25 October 2005
doi: 10.1242/jcs.02625

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

¹ School of Biological Sciences, Queen Mary, University of London, London, E1 4NS, UK
² Department of Physiology, Gower Street, University College London, London, WC1E 6BT, UK

View larger version (40K): [in a new window]   Fig. 1. Early development of the neuromuscular system in the trunk of the embryo. (A) At 16 hpf. In each spinal cord segment three or sometimes four motor neurons (RoP, MiP, CaP/VaP, as shown in green) are positioned ventrally either side of the midline (Eisen, et al., 1986). In the somite there are two populations of muscle cells, adaxial cells (shown in red) and lateral presomitic cells that differentiate into slow fibres and fast fibres respectively (Devoto et al., 1996). At around 18 hpf the motor neurons begin axon extension. Axons leave the spinal cord at a common point and grow along the medial surface of the somite (Eisen et al., 1986). Cuboidal adaxial cells, arranged in a 4x5 array adjacent to the notochord, begin to elongate and migrate within the somite (Devoto et al., 1996). (B) At 19 hpf. The leading motorneuron axon makes contact and pauses at a specialized set of muscle fibres, termed muscle pioneers. The muscle pioneers, a subset of adaxial cells that remain adjacent to the notocord, become flattened cells separating the dorsal and ventral somite. Adaxial/slow muscle fibres have formed a 1x20 array of elongated muscle fibres. (C) At 22 hpf, motor neuron axon pathways now diverge to innervate specific areas of the somite. Adaxial cells migrate radially towards the lateral surface of the somite (Devoto et al., 1996).

View larger version (24K): [in a new window]   Fig. 2. Acetylcholine-generated calcium transients in muscle fibres within the embryo. (A) The frequency of the calcium transients (mean ±s.e.m.), recorded from whole muscle fibres using Oregon Green 488 BAPTA dextran, was plotted against standard developmental time (hpf). (B) The frequency of the calcium transients (mean ±s.e.m.) measured using the high affinity Oregon Green 488 BAPTA dextran (black bars, nM range) compared with the lower affinity Fluo-4 dextran (grey bars, µM range) were plotted against standard developmental time (hpf). There was no significant difference between the frequency of the calcium signals measured with two indicators between 18 and 19 hpf, or at 21.5hpf (unpaired t-test). (C) Traces (n=13) were selected in which fluorescence changes, reported using Oregon Green BAPTA dextran, correspond to changes in cytosolic calcium ion and not to cell movements (see supplementary material Fig. S1). The duration of the signals, defined as the time taken to decay from maximum amplitude to half that value (see inset) decreased significantly between a developmental period of 18-21 hpf (Spearman Rank Correlation, r=–0.825, P<0.0001). (D) Calcium signals in the anterior trunk axial muscle display a characteristic developmental pattern between 16 and 22 hpf.

View larger version (98K): [in a new window]   Fig. 3. Expression of ryanodine receptors in embryo. (A) Phylogenetic tree of vertebrate RyR family members based on a ClustalW generated alignment of zebrafish RyR3 (CAI11683), chicken RyR3 (Q90985), human RyR3 (Q15413), mouse RyR1 (Q80X16), human RyR1 (P21817), mouse RyR2 (Q9ERN6) and human RyR2 (Q5VWP1). (B) Sequence alignment of the divergent region showing: *, identical residues;:, conservative residues;., semi-conservative residues. (C) Dorsal view of a 10 somite embryo showing expression of zfRyR3 in adaxial muscle pioneer cells of newly formed somites (arrows) and both adaxial and paraxial (arrowhead) cells of more mature somites. Anterior is to the top. (D) Transverse 15 micron section through the trunk of a 32 hpf embryo stained for zfRyR3 expression (blue). (E) Transverse 15 micron section through the trunk of a 32 hpf embryo stained with the pan muscle myosin antibody (brown) and for zfRyR3 expression (blue). Expression is detected throughout the myotome. Immunostaining in embryos aged (F) 20 hpf and (G) 24 hpf with 34C antibody (red) revealed ryanodine receptor clusters from 20 hpf onwards (arrows). Immunostaining using 34C antibody in (H) wild type and (I) nic1 zebrafish embryos at 48 hpf. Bars 10 µm.

View larger version (87K): [in a new window]   Fig. 4. Slow muscle development is disrupted in ryanodine treated embryos. Embryos were treated with ryanodine just prior to 17 hpf and fixed and stained at 24 hpf. Immunostaining with antibody F59 was performed to reveal slow muscle fibres in (A,D) control (B,E) 10 µM (C,F) 50 µM ryanodine treated embryos. Bars (A-C) 50 µm and (D,E) 10 µm. Insets (B,C) show cross section of slow muscle myosin in anterior trunk of embryos. (E,F) In ryanodine treated embryos striations (arrows with asterisks) were evident and myofibrils (arrows) that had not aligned into bundles were observed.

View larger version (72K): [in a new window]   Fig. 5. Acetylcholine generates calcium transients and regulates slow muscle development. Embryos were loaded with Oregon Green BAPTA dextran and incubated in (A) embryo medium or (B) AChR blocker -bungarotoxin (0.5 µM) for 30 minutes prior to imaging. All calcium signals were inhibited in the presence of the acetylcholine receptor blocker -bungarotoxin between 18-20 hpf (n=18). Tailcut embryos were incubated in (C) embryo medium, or (D) AChR blocker -bungarotoxin (0.5 µM) from 16 hpf and fixed at 24 hpf. Immunostaining with antibody F59 revealed slow muscle fibres. Inset shows cross section to reveal slow muscle distribution in one half of embryo. Bars 20 µm.

View larger version (68K): [in a new window]   Fig. 6. Absence of acetylcholine receptors in nic1 mutants disrupts myofibril organisation. Immunostaining with antibody F59 was performed to reveal slow muscle fibres in (A,B) 24 hpf and (C) 48 hpf wild-type embryos and (D,E) 24 hpf and (F) 48 hpf nic1 embryos. (C,F) Insets show cross section of slow muscle myosin in anterior trunk of embryo at 48 hpf. Myofibrils organisation was disrupted in the mutants (arrows). Bars (A,D) 20 µm, (B,E) 10 µm and (C,F) 50 µm. (G) Fibre length/somite width was significantly longer in mutant embryos (light-grey bars, n=22 at 24 hpf and n=13 at 48 hpf) compared with wild-type embryos (dark-grey bars, n=24 at 24 hpf and n=26 at 48 hpf; ±s.e.m., ***P<0.0001, unpaired t-test). Dual immunostaining with phalloidin and antibody F59 revealed striations in (H) wild type and (I) homozygote nic1 embryos at 48 hpf; bar 20 µm. Electron micrographs of longitudinal sections through axial muscles of the trunk of (J) wild type and (K) nic1 embryos at 48 hpf. Bars, 1 µm.