The level of LIN-14 protein forms a temporal gradient that specifies stage-specific cell lineages during development of C. elegans. Mutations that perturb LIN-14 levels perturb the temporal sequence of cell lineages. LIN-14 is a nuclear protein, but is not homologous to any known protein. To experimentally establish how graded LIN-14 levels act to specify stage-specific cell fates (including the mechanism used by
lin-14 to control downstream genes, i.e. transcription, splicing etc.) we have attempted to identify factors that mediate
lin-14 action. We expect these to include targets of
lin-14, as well as factors that act in combination with LIN-14. First, we have identified suppressors of a
lin-4(
ma161) mutation.
lin-4 is a negative regulator of
lin-14. Loss of function mutations in
lin-4 result in elevated levels of LIN-14, which imposes L1 patterns of cell lineage on post-L1 cells. Consequently,
lin-4 animals are unable to form dauers. Forty six independently isolated mutants have been identified from two screens (one million and one hundred thousand genomes mutagenized respectively), in which the dauer-defective phenotype of
lin-4 (
ma161);
daf-1(
m40) animals is suppressed. We have also isolated 10 mutants from a
lin-4;
daf-2(
e1370) strain. Of the 25 alleles tested so far, most fail to complement a
lin-14(
n179) mutation. However, 5 alleles fully complement
lin-14(
n179). One allele,
mg147, is recessive and maps to chromosome II. We are testing whether it is allelic to other known heterochronic genes on II, e.g.
lin-42(
n1089). The others (
mg148,
mg149,
mg150,
mg151) do not appear to map to either chromosome X or II. We are currently establishing the map position of these alleles. Second, we have identified candidate suppressors of
lin-14(lf) mutations. In a screen of 2 X 104 haploid genomes, eighteen mutations were identified based on their ability to suppress the sterility of
lin-14(
n179);
egl-35(
n694) double mutants. We are testing whether these suppress
lin-14(
n179) alone, and will map interesting mutations. Third, to find potential downstream targets of
lin-14, we are investigating the role of known heterochronic genes (e.g.
egl-35,
daf-12,
lin-42) in the heterochronic pathway using genetic epistasis and LIN-14 immunostaining. Fourth, we have used the yeast two hybrid system to isolate factors that interact directly with LIN-14, and have obtained interacting clones. Specific interactors include: a member of the 14-3-3 family, a myosin family member, and a novel protein. We are testing whether candidate mutations and/or overexpression of these genes result in heterochronic phenotypes, and also whether they enhance or suppress Lin-14 phenotypes.