Abstract
BackgroundAnimal models of human diseases are essential as they allow analysis of the disease process at the cellular level and can advance therapeutics by serving as a tool for drug screening and target validation. Here we report the development of a complete genetic model of spinal muscular atrophy (SMA) in the vertebrate zebrafish to complement existing zebrafish, mouse, and invertebrate models and show its utility for testing compounds that alter SMN2 splicing.ResultsThe human motoneuron disease SMA is caused by low levels, as opposed to a complete absence, of the survival motor neuron protein (SMN). To generate a true model of SMA in zebrafish, we have generated a transgenic zebrafish expressing the human SMN2 gene (hSMN2), which produces only a low amount of full-length SMN, and crossed this onto the smn-/- background. We show that human SMN2 is spliced in zebrafish as it is in humans and makes low levels of SMN protein. Moreover, we show that an antisense oligonucleotide that enhances correct hSMN2 splicing increases full-length hSMN RNA in this model. When we placed this transgene on the smn mutant background it rescued the neuromuscular presynaptic SV2 defect that occurs in smn mutants and increased their survival.ConclusionsWe have generated a transgenic fish carrying the human hSMN2 gene. This gene is spliced in fish as it is in humans and mice suggesting a conserved splicing mechanism in these vertebrates. Moreover, antisense targeting of an intronic splicing silencer site increased the amount of full length SMN generated from this transgene. Having this transgene on the smn mutant fish rescued the presynaptic defect and increased survival. This model of zebrafish SMA has all of the components of human SMA and can thus be used to understand motoneuron dysfunction in SMA, can be used as an vivo test for drugs or antisense approaches that increase full-length SMN, and can be developed for drug screening.
Highlights
Identification of the survival motoneuron gene (SMN) as the genetic cause of the motoneuron disease spinal muscular atrophy (SMA) [1] was a major advance in the motoneuron disease field
Characterization of transgenic human SMN2 gene (hSMN2) zebrafish lines To generate a complete model of SMA in zebrafish, we generated a transgenic zebrafish line expressing hSMN2
It had previously been shown that the entire human hSMN2 gene including its promoter was on a 35.5 kb BamHI fragment in the genomic clone PAC 215P15 [3]
Summary
Identification of the survival motoneuron gene (SMN) as the genetic cause of the motoneuron disease spinal muscular atrophy (SMA) [1] was a major advance in the motoneuron disease field. The result is that the vast majority (~80-90%) of SMN from the SMN2 gene lacks exon 7 (SMNΔ7) [1]. This yields an unstable protein that cannot substitute for the fulllength SMN protein [6,7,8]. We report the development of a complete genetic model of spinal muscular atrophy (SMA) in the vertebrate zebrafish to complement existing zebrafish, mouse, and invertebrate models and show its utility for testing compounds that alter SMN2 splicing
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