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ZFIN ID: ZDB-PUB-990414-44
Characterizations of the zebrafish acetylcholine receptor mutant, nic1
Sepich, D.S.
Date: 1994
Source: Ph.D. Thesis : (Thesis)
Registered Authors: Sepich, Diane
Keywords: none
MeSH Terms: none
PubMed: none
I have studied the formation of neuromuscular junctions in zebrafish to elucidate mechanisms underlying the formation of synapses. My studies have centered on the role of the nic1 gene. Zebrafish embryos mutant for the gene nic1 do not move, but otherwise develop normally. nic1 muscle fibers lack clustered acetylcholine receptors (AChRs) as revealed by binding of ligands for the AChR. nic1 fibers contract in response to direct electrical stimulation, but unlike wild-type, are not depolarized in response to an ACh agonist. Thus, there is no evidence of clustered or dispersed AChRs. These results showed that although myofibers in nic1 mutants are mechanically intact, and motoneurons form synapses, the nic1 mutation blocks the expression and clustering of functional AChRs in the muscle. I investigated whether nic1 effects signaling between motoneurons and muscles that regulates AChR clustering. Robert Ho and I made mosaic embryos by transplanting precursor cells from wild-type to mutant embryos. nic1 muscle cells failed to cluster AChRs even when contacted by wild-type motoneurons, whereas nic1 motoneurons induced AChR clustering on wild-type muscle. Additionally, I explored the intrinsic ability of nic1 muscle cells to cluster AChRs in culture. Mutant muscle cells failed to cluster AChRs under culture conditions that supported clustering on wild-type cells. These results showed that the nic1 mutation acts autonomously in muscle cells rather than by affecting signaling between motoneuron and muscle required for synapse formation; the wild-type nic1 gene is necessary in muscle for expression and clustering of AChRs. My analysis of nic1 suggests that nic1 affects the AChR $alpha$-subunit gene. I showed that the nic1 $alpha$-subunit gene lacks part of intron 6 which may include where the branchpoint forms during splicing. I hypothesize that the deletion results in decreased splicing efficiency of this intron. I found that the $alpha$-AChR mRNA is larger in nic1 embryos and abnormally accumulated within the muscle cell. J. Wegner and I rescued the clustering defect by providing wild-type $alpha$-subunit mRNA. My analysis of nic1 suggests that inefficient splicing of this small intron blocks assembly of AChRs leading to paralysis.
Ph.D. Thesis, University of Oregon