Neuromuscular synaptogenesis in wild-type and mutant zebrafish

Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, 3610 Hamilton Walk, Philadelphia, PA 19104-6074, USA.
Developmental Biology (Impact Factor: 3.55). 10/2005; 285(2):340-57. DOI: 10.1016/j.ydbio.2005.06.027
Source: PubMed


Genetic screens for synaptogenesis mutants have been performed in many organisms, but few if any have simultaneously screened for defects in pre- and postsynaptic specializations. Here, we report the results of a small-scale genetic screen, the first in vertebrates, for defects in synaptogenesis. Using zebrafish as a model system, we identified seven mutants that affect different aspects of neuromuscular synapse formation. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Characterization of mutant and wild-type zebrafish, from the time that motor axons first arrive at target muscles through adulthood, has provided the new information about the cellular events that occur during neuromuscular synaptogenesis. These include insights into the formation and dispersal of prepatterned AChR clusters, the relationship between motor axon elongation and synapse size, and the development of precise appositions between presynaptic clusters of synaptic vesicles in nerve terminals and postsynaptic receptor clusters. In addition, we show that the mechanisms underlying synapse formation within the myotomal muscle itself are largely independent of those that underlie synapse formation at myotendinous junctions and that the outgrowth of secondary motor axons requires at least one cue not necessary for the outgrowth of primary motor axons, while other cues are required for both. One-third of the mutants identified in this screen did not have impaired motility, suggesting that many genes involved in neuromuscular synaptogenesis were missed in large scale motility-based screens. Identification of the underlying genetic defects in these mutants will extend our understanding of the cellular and molecular mechanisms that underlie the formation and function of neuromuscular and other synapses.

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Available from: Roland Dosch
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    • "Subsequently we examined the number and spatial distribution of NMJs with SynaptcountJ, a plugin for ImageJ that permits quantification of synapse number (see methods). Analysis was restricted to the second day of development (Figure 1A) because by this stage a small, easily quantifiable network of arbours has been established in each somitic region ([48]; Figure 1B). Since our previous work suggested that NO/cGMP signalling affects motor axon branch formation without impairing motor root growth, we segregated puncta into two domains: those located on the motor axon fascicle and those located on motor axon branches (Figure 1A). "
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    • "As previously described, neuromuscular defects are apparent in ALS rodent models and patients well before symptom onset and MN loss [3,4]; therefore, we next performed a detailed pathological characterization of NMJs and MNs in control and transgenic G93A-SOD1-GFP zebrafish. Techniques to quantify NMJ innervation by MNs are well-established in rodents [25,45,50,51] and larval zebrafish [52-55], however, only limited information regarding visualization and quantification of juvenile and adult zebrafish NMJ integrity are available [32,56]. For the current studies, we labeled presynaptic MNs and postsynaptic AChR clusters with SV2/NF and αBTX, respectively, and quantified the number of intact NMJs in control AB and transgenic G93A-SOD1-GFP zebrafish between 10 and 60 weeks of age (Figure 4). "
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    • "Thus, the NMJ is an attractive model for studying the sequence of events during synapse assembly. Furthermore, because myotubes and motor neurons develop in a rostral-to-caudal progression, the whole sequence of events underlying NMJ assembly can be viewed in a single zebrafish embryo (Flanagan-Steet et al., 2005; Panzer et al., 2005, 2006). Two time lapse imaging studies have demonstrated that prepatterned AChRs exist on muscle fibers before the arrival of motor axons (Flanagan-Steet et al., 2005; Panzer et al., 2006). "
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