GFP screens for regulators of motor neuron differentiation.

With its well-defined nervous system and facile genetics, C. elegans is a powerful model system in which to identify molecules that specify motor neuron fate. A-type (DA,VA) and B-type (DB,VB) motor neurons in the nematode ventral nerve cord are required for normal locomotion. A-type motor neuron differentiation depends on the cell autonomous activity of the UNC-4 homeoprotein and its transcriptional co-repressor, UNC-37. unc-4 mutants are unable to execute normal backward movement due to the miswiring of VA motor neurons with VB-type synaptic inputs. In addition, A-type motor neurons express B motor neuron GFP transgenes in unc-4 and unc-37 mutant backgrounds. We believe the unc-4 miswiring defect is due to a limited change in cell fate -- i.e. VA motor neurons now assume some of the characteristics of their VB sister cells. Thus, unc-4 target genes may specify B motor neuron fate. In an effort to identify these genes, we have conducted a visual screen for mutations affecting the expression of acr-5::YFP, a B motor neuron marker that is ectopically expressed in A motor neurons in unc-4 mutants. A clonal screen of 2200 EMS mutagenized F1 worms revealed five mutants with reduced acr-5::YFP expression in the ventral nerve cord but which have normal acr-5::YFP expression in other neurons in head and tail ganglia. One isolate contains two recessive alleles, wd72 and wd73, that map to separate unlinked loci. Together, wd72 and wd73 confer a forward movement defect resembling that of vab-7, a transcription factor that specifies DB fate. wd72 alone results in reduced ventral cord acr-5::YFP expression but does not perturb locomotion. Neither wd72 nor wd73 affect expression of GFP markers for other classes of ventral cord motor neurons (e.g. unc-4::GFP, A-type; lin-11::GFP, VC). We hypothesize that wd72 and wd73 may perturb genes with partially redundant functions during DB motor neuron differentiation. In a second genetic experiment, we used the Union Biometrica COPAS Biosort to screen the progeny of 6400 F1 animals. This machine is capable of sorting worms by size (i.e, age) and fluorescence. Fourteen mutations with reduced acr-5::YFP expression in the ventral nerve cord were isolated. A careful genetic and phenotypic analysis of these mutations is expected to reveal genes that regulate B-class motor neuron fate.