Heterochronic genes regulate the timing of cell fate determination. Mutations in heterochronic genes cause alterations of cell fates in which stage-specific events are either omitted or reiterated, resulting in precocious or retarded phenotyes respectively. LIN-14 is a key heterochronic regulatory factor that promotes early larval fates in C. elegans. LIN-14 protein decreases over time and specifies the timing of the L1 to L2 cell lineage transition. Mutations that perturb the level of LIN-14 protein perturb the temporal sequence of cell lineages during development. LIN-14 is a novel nuclear protein and the method by which LIN-14 acts to specify stage-specific cell fates is unknown. To elucidate the role of
lin-14 in heterochronic control of development, we are identifying factors that mediate
lin-14 action as genetic suppressors or enhancers of
lin-14 mutant phenotypes. To identify genes negatively regulated by
lin-14 , we performed a screen for suppressors of the heterochronic phenotypes conferred by a
lin-14(lf) mutation. Preliminary screening of 4.4 x 10 4 haploid genomes produced 36 mutants in at least three complementation groups: Class 1: 2 alleles of
let-7 X; Class 2: 8 alleles of a gene that we have tentatively called
sol-1 ( s uppressor o f l in-14 ) ; Class 3: 26 unmapped alleles (Non-
let-7 ). This work focuses primarily on the Class 2
sol-1 alleles.
sol-1 mutations cause a weak retarded heterochronic phenotype when not in the presence of decreased
lin-14 gene activity. Based on genetic mapping and complementation tests we are currently determining, whether
sol-1 corresponds to a known gene or defines a new one. That the
sol-1 phenotype is unlike any of the published heterochronic mutants suggests that we have identified a new heterochronic gene.
egl-35 mutations synthetically enhance the precocious phenotype of
lin-14 mutations. This synthetic enhancement with the two alleles suggests that
lin-14 and
egl-35 genes interact in the same pathway and that this interaction is a positive one. We are characterizing the
egl-35 phenotype in more detail and intend to fine map and clone
egl-35 . The C. elegans timeless homologue,
tim-1, maps to the
egl-35 genetic region.
tim-1 is a good candidate for
egl-35 because another heterochronic gene,
lin-42 , which also enhances a
lin-14 phenotype encodes the C. elegans period homologue ( period and timeless interact to control circadian timing in flies and mammals). We are attempting to rescue the
egl-35 phenotype with genomic DNA spanning the timeless gene, and we are also sequencing the timeless gene from
egl-35 alleles to identify potential lesions. We are testing the hypothesis that
tim-1 is a heterochronic gene by performing RNAi with
tim-1 and asking if this leads to a heterochronic phenotype. We are also testing if RNAi of
tim-1 enhances a
lin-14 phenotype, as would be expected if
tim-1 is
egl-35 . We are investigating whether other C. elegans homologs of circadian genes have a function in the heterochronic pathway. We are doing RNAi with C. elegans ORFs that have sequence similarity to circadian genes and evaluating the effect of RNAi on the phenotypes of key heterochronic pathway regulators, such as C. elegans
lin-14 ,
lin-28 ,
let-7 , etc. In preliminary experiments, we have found that RNAi with the C. elegans homolog of the circadian gene doubletime suppresses the mutant (lf) phenotype of the heterochronic gene
let-7 .