lin-57 was identified in a screen for heterochronic mutants that exploits the adult specific nature of the
rol-1 phenotype.
rol-1(
e61) mutants exhibit a stage-specific locomotion defect: larvae swim in the normal sinusoidal pattern but adults roll in circles. This adult specific
rol-1 phenotype depends upon the synthesis of adult cuticle and is temporally altered by mutations in the heterochronic gene pathway. For example,
lin-4 and
lin-29 mutants never synthesize adult cuticle and consequently
lin-4 rol-1 and
rol-1 lin-29 double mutants never roll. Conversely,
lin-14 and
lin-28 mutants synthesize adult cuticle early and
rol-1 double mutant combinations with these genes result in animals that roll as larvae. In attempts to identify additional heterochronic genes, we mutagenized
rol-1(
e91) animals and screened for animals that either roll precociously or fail to roll as adults. Among the mutants identified in this screen was a precocious roller that defined
lin-57. In
lin-57 mutants, seam cell terminal differentiation and adult cuticle synthesis occur one stage early, during the L3 molt. To better understand the role of
lin-57 in the heterochronic gene pathway, we cloned it. We found that
lin-57 is
hbl-1, a gene identified based on high sequence similarity to the Hunchback gene of Drosophila (Fay et al. 1999). We observe a precocious heterochronic defect in animals injected with double stranded RNA (RNAi) using a portion of the
hbl-1 cDNA. Furthermore, a five base pair deletion that alters the
exon-2 splice donor sequence was identified in one
lin-57 allele. Interestingly, two non-rescuing
lin-57::gfp constructs (kindly provided by D. Fay) with different 3' UTRs showed different temporal expression patterns. A
lin-57::gfp construct containing
unc-54 3'UTR is expressed at all stages. In contrast, a
lin-57::gfp construct containing the
lin-57 3'UTR is excluded from adults. Examination of the
lin-57 3'UTR reveals five putative binding sites for
let-7, a member of the heterochronic gene pathway shown to encode a small regulatory RNA (Reinhart et al. 2000) .
let-7 mutants exhibit a retarded heterochronic defect, consistent with a model where
let-7 RNA acts as a
lin-57 repressor. We are in the process of conducting genetic and molecular experiments to test this model. Reinhart B.J., Slack F.J., Basson M,, Pasquinelli A.E., Bettinger J.C., Rougvie A.E., Horvitz H.R. and Ruvkun G. (2000) The 21-nucleotide
let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901-6. Fay D.S., Stanley H.M., Han M. and Wood W.B. (1999) A Caenorhabditis elegans homologue of hunchback is required for late stages of development but not early embryonic patterning. Dev Biol 205:240-53.