Cloning and characterization of the sympathetic nervous system mutant, Sym3, in zebrafish

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9541 Background: Neuroblastoma (NB) is a neural crest tumor manifesting in the adrenal gland and the sympathetic ganglia. The goal of this project was to use the zebrafish model to identify genes involved in the development of the peripheral sympathetic nervous system (PSNS). We used the expression of tyrosine hydroxylase (TH) in the cervical sympathetic ganglion (cervical complex, CC), as a marker for PSNS development. TH expression can be observed by whole-mount RNA in situ hybridization in 2–3-day zebrafish embryos in the CC. Methods: In an ethyl nitrosourea mutagenesis screen for mutations that affect PSNS development, we identified a putative mutant, Sym3, showing a change in modeling of the TH staining cells in the region of the CC. Results and Conclusion: The TH-positive cells were scattered rather than conglomerated together as is typical of cells in the normal CC. The mutant was also characterized by a “curly-tail” phenotype. After the mutation was recovered from the F3 generation, heterozygous pairs harboring the mutation were identified. Using genome wide scanning PCR assay, linkage of the Sym3 mutation to microsatellite markers z9704 and z1351, located on Linkage Group 1, was established. Complementation assays were performed with other known “curly tail” mutants in the genetic interval, which revealed that Sym3 and a previously described vic mutant were in the same complementation group. The vic mutant is characterized by curved body axis, left to right asymmetry and kidney cysts and results from a mutation in the ARL13b gene, a protein of the Ras superfamily involved in intracellular trafficking and cilial motility. The vic mutation is a C to A transition at position 104 in the second exon of the open reading frame (T35L). The ARL13b gene in Sym3 has a four aa insertion at the 5’ end of the 4th exon of the coding region.The mutant Sym3 phenotype can be rescued by overexpression of the normal ARL13b gene. The vic mutant showed defective cilia formation in the kidney. However, immunocytochemistry showed normal cilia in the Sym3 mutant, suggesting that the Sym3 allele of ARL13b affects the development of alternative tissues during development, such as the aggregation of sympathetic neurons into discrete ganglia. Further functional characterization of the Sym3 mutation is ongoing. No significant financial relationships to disclose.