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.


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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    ABSTRACT: Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.
    PLoS ONE 01/2014; 9(1):e86930. DOI:10.1371/journal.pone.0086930 · 3.23 Impact Factor
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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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    ABSTRACT: Background Amyotrophic lateral sclerosis (ALS) is a fatal disorder involving the degeneration and loss of motor neurons. The mechanisms of motor neuron loss in ALS are unknown and there are no effective treatments. Defects in the distal axon and at the neuromuscular junction are early events in the disease course, and zebrafish provide a promising in vivo system to examine cellular mechanisms and treatments for these events in ALS pathogenesis. Results We demonstrate that transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in early defects in motor neuron outgrowth and axonal branching. This is consistent with previous reports on motor neuron axonal defects associated with familial ALS genes following knockdown or mutant protein overexpression. We also demonstrate that upregulation of growth factor signaling is capable of rescuing these early defects, validating the potential of the model for therapeutic discovery. We generated stable transgenic zebrafish lines expressing G93A-SOD1 to further characterize the consequences of G93A-SOD1 expression on neuromuscular pathology and disease progression. Behavioral monitoring reveals evidence of motor dysfunction and decreased activity in transgenic ALS zebrafish. Examination of neuromuscular and neuronal pathology throughout the disease course reveals a loss of neuromuscular junctions and alterations in motor neuron innervations patterns with disease progression. Finally, motor neuron cell loss is evident later in the disease. Conclusions This sequence of events reflects the stepwise mechanisms of degeneration in ALS, and provides a novel model for mechanistic discovery and therapeutic development for neuromuscular degeneration in ALS.
    Molecular Neurodegeneration 08/2012; 7(1):44. DOI:10.1186/1750-1326-7-44 · 6.56 Impact Factor
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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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    ABSTRACT: The study of nervous system development has been greatly facilitated by recent advances in molecular biology and imaging techniques. These approaches are perfectly suited to young transparent zebrafish where they have allowed direct observation of neural circuit assembly in vivo. In this review we will highlight a number of key studies that have applied optical and genetic techniques in zebrafish to address questions relating to axonal and dendritic arbor development,synapse assembly and neural plasticity. These studies have revealed novel cellular phenomena and modes of growth that may reflect general principles governing the assembly of neural circuits.
    Developmental Neurobiology 03/2012; 72(3):346-57. DOI:10.1002/dneu.20874 · 3.37 Impact Factor
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