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First published online 5 May 2004
doi: 10.1242/dev.01123

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Increased neuromuscular activity causes axonal defects and muscular degeneration

¹ Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA
² Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York, NY 11794, USA

View larger version (105K): [in a new window]   Fig. 1. twister mutants display aberrant motor axon trajectories. (A) The three primary axons, CaP, RoP and MiP, initially share a path from the spinal cord to the choice point, at the level of the horizontal myoseptum (broken line). (B-D) Wild-type (26 hpf) and twister mutant embryos stained with znp-1 antibody (black line indicates the ventral aspect of the spinal cord, white line the level of the choice point). (B) In wild-type embryos, axons migrate to the choice point, from which the CaP axon migrates into the ventral myotome (white arrow), whereas the MiP projects a collateral into the dorsal myotome (white arrowhead). (C) In heterozygous twister mutant embryos, primary motor axons develop ectopic branches at the region of the choice point (black arrows). (D) In twister homozygous mutant embryos, motor axons form ectopic branches at the choice point (black arrow) or in the ventral myotome (yellow arrows). In addition, motor axons are stalled along the common path (blue arrows; all lateral views).

View larger version (112K): [in a new window]   Fig. 2. In twister mutant embryos, slow and fast muscle fiber development is severely affected. Confocal micrographs of 26 hpf wild-type and mutant embryos stained with antibodies specific for slow muscle fibers (F59; A,C,E) or fast muscle fibers (F310; B,D,F; all lateral views). (A) In wild-type embryos, slow muscle forms a superficial monolayer of striated fibers arranged in parallel. (B) In wild-type embryos, fast muscle fibers are oriented obliquely and form the bulk of the myotome. (C,D) In twister heterozygous mutants, individual fibers are thinner, such that gaps appear between them (arrowheads), and somite boundaries are irregular (arrow). (E,F) twister homozygous mutants exhibit severely disrupted slow and fast myofiber organization. Some fibers have detached from the somite boundaries, which are irregular (white arrow). Many muscle fibers have contraction clots of myofibril material, as shown by densely stained clots (arrowheads). Scale bar: 50 µm.

View larger version (170K): [in a new window]   Fig. 3. Muscle cells exhibit ultrastructural defects in twister mutant embryos. Electron micrographs of cross-sections (A,C,E) and longitudinal sections (B,D,F) of somitic muscle taken from comparable rostro-caudal levels of 26 hpf wild-type and twister mutant embryos. (A) Cross-section of wild-type muscle with mature, ordered myofibrils. Polyhedral-shaped myofibrils (open arrowheads) are surrounded by an organized membrane system of tubules and sarcoplasmic reticulum cisternae (black arrow). Myofibrils form a characteristic double-hexagonal array of thick and thin filaments at the A band (open arrowheads and inset), and a tetragonal arrangement of thin filaments in proximity of the Z line (black arrowhead). (B) Longitudinal section of wild-type muscle. Myofibrils are organized into sarcomeres with prominent A (A) and I (I) bands. Triads (3 arrows) are associated with the Z (z) lines. (C) Cross-section of heterozygous twister mutant muscle in which thick filaments are frequently surrounded by more than six thin filaments (open arrowhead and inset). Similarly, the surrounding membrane system (T tubules and sarcoplasmic reticulum) is less organized (black arrows). (D) Longitudinal sections of heterozygous mutant muscle show ill-defined A (A) and I (I) bands and Z (z) lines. (E) Cross-section of homozygous twister mutant muscle. Myofibrils are in disarray and extend in various orientations (double arrows), and the membrane system is poorly developed. (F) In longitudinal sections, myofibril bundles are thin and misaligned. A primitive T-tubule is present (3 arrows), but the typical triad organization of SR cisternae and T-tubules is absent.

View larger version (20K): [in a new window]   Fig. 4. twister-related nerve and muscle defects are caused by prolonged synaptic transmission. (A) Injecting buffer into embryos obtained from two twister heterozygotes resulted in the expected distribution of axonal and muscle phenotypes, i.e. 75% displayed wild-type or heterozygous axonal and myofiber phenotypes (white bar), whereas 25% displayed severe axonal and myofiber defects (gray bar; n=261, from three experiments). In contrast, -BTX injection increased the proportion of embryos displaying wild-type or heterozygous axonal and myofiber morphology (88.1±3.2%), and decreased the proportion of embryos displaying twister homozygous defects (11.9±3.2%; n=475 from six experiments; P<0.0001 for Fisher's exact test). (B-G) Comparison of spontaneous (mEPC) and evoked (EPC) synaptic currents obtained by whole-cell voltage clamp of 72 hpf wild-type (B) and heterozygous twister (C) larvae. (B,C) The plot represents the average of 10 individual spontaneous currents, aligned at the peaks. (D,E) Frequency histograms of decay times for individual mEPCs from wild-type (D) and heterozygous twister (E) muscle. Decay times were determined on the basis of 90% decay from peak amplitude. Note that in wild-type muscle, all events showed decay times shorter or equal to 10 mseconds, whereas in mutant muscle most of the events had decay times longer than 10 mseconds (black arrows). (F,G) Evoked end-plate currents obtained from wild-type (F) and heterozygous twister (G) muscle in response to 50 Hz stimulation of the spinal cord. Ten consecutive trains were averaged. Time points of stimulation are indicated by filled circles and the broken line indicates the baseline holding current.

View larger version (32K): [in a new window]   Fig. 5. twister mutation is a point mutation in the M2 domain of chrna1. (A-C) Molecular and genetic mapping of twister locus to LG6. (A) PCR amplification using SSLP marker z6601 on DNA obtained from the two grandparents (G0) and from pools of F2 homozygous mutants, heterozygous mutants and wild-type siblings, respectively. The shorter PCR fragment from the G0 Tüebingen heterozygote segregates with the homozygous mutant pool, from which the longer, WIK-specific allele is absent. (B) PCR amplification of DNA obtained from individual mutant embryos with marker z6601. Recombinant embryos (R) were detected by the presence of a larger WIK-specific PCR fragment. (C) The twister locus maps between markers z6601 and z9739. (D) Sequence analysis of chrna1 in twister revealed a single nucleotide change, TC, giving rise to a leucine to proline amino acid substitution at codon 258. (E) The L258P mutation is located in the second transmembrane (M2) domain, which contributes to the cation-selective channel pore. (F) Cross-species sequence alignment of the CHRNA1 M2 domain. The leucine 258 residue affected in twister mutants is 100% conserved across species. GenBank Accession numbers are in parentheses. (G) Whole-cell recordings of spontaneous synaptic currents from nic-1b107 homozygous fish expressing receptors containing either wild-type -subunits or the L258P mutant subunits. The holding potential was –90 mV and the vertical calibration corresponds to 20 pA for wild-type and 60 pA for L258P mutant. (H) The distribution of mean values for exponentially fitted current decays from 8 muscles expressing wild-type and 9 mutant twister -subunit receptors. Between 6 and 276 synaptic events were used to compute each mean value (overall mean and standard errors are indicated).

View larger version (117K): [in a new window]   Fig. 6. Excessive postsynaptic activity modulates axon growth and synapse formation. Confocal analysis of 17 hpf wild-type and nic1twister dbn12 mutant embryos, stained with antibody F59 specific for slow muscle fibers (A-C), or double stained with motoneuron-specific antibodies (D,G,J,M-O), and with AlexaFluor 594-conjugated -BTX to visualized clustered AChRs (E,H,K,M-O) at 17 hpf (A-L) or 26 hpf (M-O; all lateral views). (A,B) In 17 hpf wild-type and nic1twister dbn12 heterozygous mutant embryos, muscle fibers are thin and elongated but few display striations. (C) In nic1twister dbn12 homozygous mutant embryos, muscle fibers are less elongated with no visible striations. In homozygous mutant embryos, somites are compressed along the anterior-posterior axis and expanded along the dorso-ventral axis. (D-F) In 17 hpf wild-type embryos, motor axons have reached or just extended past the choice point (the level is indicated by white bars). Note the elaborate and fan-like morphology of the axonal tip, characteristic of advancing growth cones (white double arrowhead). As motor axons pioneer into the somites, clustered AChRs emerge and colocalize along the extending axon, reminiscent of en passant synaptic contacts. These dense AChR clusters decorate parts of the axon, except for the presumptive growth cone (white arrow) which precedes the distal limit of clustered AChR (white arrowhead). (G-I) In heterozygous embryos, most motor axons extend normally and their presumptive growth cones display a wild-type-like morphology (white arrow). However, a significant fraction of heterozygous motor axons stall before reaching the choice point, and presumptive growth cones appear smaller and less elaborate (red arrow). On those axons, dense AChRs clusters are localized distal to presumptive growth cones (white arrow). Note that the morphology of these aneural AChR clusters is indistinguishable from those that co-localize with the axon. (J-L) In nic1twister dbn12 homozygous mutant embryos, many motor axons (red arrows) are stalled before the choice point. Growth cones are less elaborate and appear collapsed (white double arrowhead), and AChR clusters are smaller and scattered throughout the somite (white arrowheads). (M) In 26 hpf wild-type embryos, AChR clusters are restricted along the lengths of the ventral and dorsal motor axons (yellow arrows) and along the somite boundaries (yellow arrowhead). (N) In nic1twister dbn12 heterozygous embryos, AChR clusters co-localize along the lengths of the ventral and dorsal axons (yellow arrows) and along the somite boundary (yellow arrowhead). AChR clusters are also detected along aberrant branches (blue arrowhead). (O) In 26-hpf nic1twister dbn12 homozygous mutants, smaller and fewer AChR cluster co-localize with axonal branches (blue arrowhead). Somite boundary localization of clustered AChRs is strongly reduced. Scale bar: 50 µm.