The heterochronic gene
lin-14 controls the timing of post-embryonic developmental events, and perturbations in
lin-14 gene activity result in the inappropriate timing of stage specific events. Genetic evidence suggests two gene activities for
lin-14,
lin-14a for proper L1 timing and
lin-14b for L2. In addition, the
lin-14 gene uses two promoters, A and B, to produce three distinct proteins (A, B1, and B2) by differential splicing of the transcript from the B promoter. We have investigated the possibility that the different proteins are the basis for the
lin-14 genetic activities. The
lin-14 gene structure and all three transcripts are conserved in C. briggsae. Sequencing loss-of-function mutations has revealed that while null mutations map to a conserved region common to all products, mutations which only effect the
lin-14b genetic activity are specific to LIN-14B1 and LIN-14B2. This suggests that LIN-14B1 and LIN-14B2 are necessary for proper L2 timing, and LIN-14A is sufficient for L1 fates. A single
lin-14a mutation that has been identified is a point mutation common to all three isoforms. We are currently investigating the rescuing activity of genomic constructs with LIN-14A deleted to determine the necessity of LIN-14A and the sufficiency of LIN-14B. We have examined the possibility that an expression difference from the promoters, rather than biochemical differences among the proteins, is responsible for the
lin-14 activities. For example, LIN-14A may be expressed early to allow L1 fates and LIN-14B may be expressed later to allow L2 fates. Two
lin-14b mutations are
lin-14b nulls,
lin-14(
n355n534amber) and
lin-14(
n360), a gamma allele. Neither mutant produces LIN-14B1 or B2 by Western blots with exon-specific antibodies. In both mutants, immunostaining detected LIN-14A protein in all cell types previously determined to express LIN-14. Subsequently, we examined these mutants using
lin-4 and
lin-14(
n355gf) which disrupt
lin-4 downregulation of LIN-14 and allow a later expression of LIN-14A.
lin-14(
n355n534amber) lacks L2 lineages although LIN-14A is expressed at all stages of development. However, the opposite is suggested by
lin-4(
e912);
lin-14(
n360), which appears to have wild-type timing. Western analysis and antibody staining suggest this disparity may be due to overall lower LIN-14A expression in
lin-14(
n355n534) and that LIN-14A can be sufficient for all
lin-14 function as seen in
lin-4(
e912);
lin-14(
n360). Another possibility is that
lin-4(
e912) is suppressing the
lin-14 phenotype via another gene, such as
lin-28. We are currently analyzing
lin-4(
e912);
lin-14(
n355n534) to verify that
lin-4 is acting directly through
lin-14.