Supplemental 2: fsn-1 is necessary and sufficient in presynaptic neurons

1. P0 wild-type fsn-1 like AB P1 ABp ABa EMS P2 P3 E MS C ABpr ABar ABpl ABal Motoneurons (Presynaptic for NMJs) Muscle (Postsynaptic for NMJs) Supplemental 2: fsn-1 is necessary and sufficient in presynaptic neurons a Mosaic analysis: fsn-1 is required in presynaptic motoneurons, but not post-synaptic muscle cells at GABAergic NMJs. InC. elegans,the motoneuron lineage (AB) separates from the muscle lineage (P1) in the first cell division. Eight out of eight mosaic animals expressing FSN-1(+) only in the AB lineage were wild-type for juIs1 synaptic marker. All twelve animals that express FSN-1(+) only in the P1 lineage displayed fsn-1-like juIs1 defects. Each circle represents an individual mosaic animal that expressed the FSN-1(+) array in the indicated lineage. Filled circles indicate wild-type phenotype and the clear ones fsn-1 mutant phenotype. Methods: Neuron and muscle cells have morphologically distinct nuclei that can be identified by DIC optics or nuclei GFP markers. We generated a fsn-1; juIs1 transgenic line carrying an extrachromosomal array by co-injecting the FSN-1 genomic clone and SUR-5::GFP nuclei marker1. The extrachromosomal array is subject to random loss during somatic cell divisions. SUR-5::GFP marker expresses GFP in the nuclei of the cells retaining the array, allowing us to identify mosaic animals. Mosaic animals that retained FSN-1(+) only in AB lineage, or P1 lineage were identified from the transgenic lines and individually scored for their juIs1 phenotype. Expression of FSN-1 in GABAergic motoneurons is necessary and sufficient to rescue juIs1 phenotypes b b1 b1: juIs1 synaptobrevin::GFP synaptic vesicle marker. Puncta (1-2 m) represent individual GABAergic NMJs. b2 b2: synaptic defects of fsn-1; juIs1 mutants include clusters of puncta and bigger gaps b3 b3: fsn-1 defects fully rescued by pJH182, an unc-25 promoter (GABAergic motoneuron specific) driven FSN-1 cDNA clone b4 b4: No rescue of synaptic defects by pJH113, a myo-3 promoter (muscle-specific) driven FSN-1 cDNA clone. 1. Yochem, J., Gu, T., and Han, M. Genetics 149:1323-1334 (1998)