The goal of this proposal is to isolate vertebrate genes that play a role in neuromuscular synapse formation and maintenance, using zebrafish as a model system. Previous fish mutagenesis screens have not focused on mutants that affect neuromuscular synaptogenesis, in part because these synapses need to be labeled with antibodies or toxins that specifically label different synaptic components and visualized using light microscopy at relatively high magnification. Over the last year, my lab has participated in a pilot mutagenesis screen conducted by Drs. Mary Mullins and Michael Granato in the Dept. of Cell and Developmental Biology at the University of Pennsylvania. My lab developed an assay for neuromuscular synapses in zebrafish utilizing antibodies against synaptic vesicles to mark presynaptic terminals, fluorescent conjugated alpha-bungarotoxin to label acetylcholine receptor (AChR) clusters, and high resolution fluorescence microscopy in intact fish at 48 hours post fertilization (hpf). Preliminary results demonstrate that we have identified several mutants with defects in different aspects of neuromuscular synaptogenesis at 48 hpf, and that some of these mutants also have motility defects. These mutants fall into three overlapping categories: aberrant synapse formation (too many, too few or mislocalized pre- and/or postsynaptic specializations); normal synapse formation, followed by synapse loss and/or redistribution; and aberrant primary and/or secondary motor axon branching within body wall musculature, resulting in aberrant endplate bands within individual muscles. Based on our success with this small, pilot screen, we propose to first, define the primary defect in 2-3 of the isolated mutants by analyzing synaptic structure and function; second, to determine the genetic map position of mutated genes for 2-3 mutants using complementation, mapping using an established set of molecular markers, and linkage analyses; and third, to isolate new mutations in genes required for neuromuscular synapse formation and maintenance by continuing and expanding our screen of mutant fish. Taken together, these approaches will allow us to study the genetic, molecular and cellular mechanisms of these processes in vertebrates. This R21 proposal will allow us to use mutagenesis in zebrafish to identify some of the genes required for neuromuscular synapse formation and maintenance, and expand the repertoire of tools available in my lab to address these fundamental questions in zebrafish and mice in the future.
| Panzer, Jessica A; Song, Yuanquan; Balice-Gordon, Rita J (2006) In vivo imaging of preferential motor axon outgrowth to and synaptogenesis at prepatterned acetylcholine receptor clusters in embryonic zebrafish skeletal muscle. J Neurosci 26:934-47 |
| Panzer, Jessica A; Gibbs, Sarah M; Dosch, Roland et al. (2005) Neuromuscular synaptogenesis in wild-type and mutant zebrafish. Dev Biol 285:340-57 |
