Fluorescence was observed at the end of the L1 stage in transgenic worms that express GFP fused to the pri-
let-7B start site. Detection of GFP mRNA, driven by both
let-7 promoter A and B sequences in the transgenic worms, mirrored that of endogenous
let-7 primary transcripts, indicating that expression of
let-7 is repressed largely at the transcriptional level from embryogenesis until the late L1 stage. Consistent with the reporter analysis, pri-
let-7 was first observed during the late L1 stage. We detected all three pri-
let-7 isoforms, and coordinate expression of these isoforms oscillated throughout development. The low levels of pri-
let-7 at most mid-larval time points and the slight shifts in the timing of pri-
let-7 expression between experiments indicate that expression of endogenous pri-
let-7 transcripts is dynamic, and that even slight changes in culture conditions can affect the rate of development and thus pri-
let-7 expression. GFP mRNA levels of the
let-7 promoter reporter oscillated with a frequency identical to that of endogenous pri-
let-7 expression, suggesting that transcriptional mechanisms largely control the cycling pattern of pri-
let-7 expression. Consistent with previous reports, pre- and mature
let-7 RNAs were undetectable until the L3 stage. In the L3 and L4 stages, levels of pre-
let-7 oscillated in parallel to those of pri-
let-7, while mature
let-7 accumulated to a relatively constant level.