Development and analysis of a Zebrafish model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by a loss of alpha-motoneurons in the spinal cord. SMA is caused by low levels of the ubiquitously expressed survival motor neuron (SMN) protein. To mimic SMA in zebrafish, antisense morpholino oligonucleotides have been utilized to reduce Smn levels in the developing embryo. When Smn levels are reduced throughout the entire embryo, motor axon pathfinding defects are observed. These defects are specific to motor axons; other sensory and interneuron axons were unaffected by Smn knockdown. Reduction of Smn in individual motoneurons revealed that smn is acting cell-autonomously. These results show that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss. A paramount question in SMA research is why reduced levels of SMN lead to a motoneuron-specific disease. It has been hypothesized that SMN may have a dual function: a well-characterized role in mediating snRNP assembly and a novel motor axonal-specific function. To begin to identify which function is important for axonal outgrowth and potentially SMA, non-Smn components in these separate pathways have been knocked-down by morpholino in zebrafish. Knockdown of Gemin2, a Smn interacting snRNP assembly component, does not yield motor axon defects suggesting a non-snRNP assembly function for Smn in motor axon outgrowth. A genetic model of SMA utilizing a zebrafish smn mutation can address questions that morpholinos alone cannot. Because targeted mutagenesis is unavailable in zebrafish, rapid high-throughput screens for mutations in a particular gene of interest are necessary to further study gene function. Screening methods have been developed to identify both ENU-induced point mutations and gamma-induced deletion mutations in the zebrafish smn gene. Targeted Induced Lesion IN Genomes (TILLING) has identified a mutation in the smn coding region that may affect gene function. A mutation in the smn gene will allow for development of a genetic zebrafish model of SMA and further elucidation of the neuropathology and etiology of the disease.