During early larval development, a cascade of signaling molecules directs the decision to either promote reproductive growth or form the alternative dauer larvae. In the dauer formation genetic pathway, two branches converge on
daf-12 and a third branch interacts with
daf-12 . To initiate molecular studies on the regulation of
daf-12 function, we performed Northern analysis of mRNA from daf mutant strains that were grown under dauer inducing conditions. The mRNA was hybridized with a segment of the
daf-12 gene that is shared by the three transcripts. We observed a decline in steady-state mRNA levels for each of the three
daf-12 isoforms in
daf-3 ,
daf-7 and
daf-16 compared to wild type animals. Thus,
daf-12 mRNA levels are regulated by TGF-ß and insulin-like signaling components of the dauer pathway. The molecular analysis of the
daf-12 cDNAs has shown three transcripts of different lengths due to differential splicing, termed isoforms A1, A2 and B. Northern analysis showed that the
daf-12 isoforms are expressed throughout development. It is possible that the three isoforms function in different tissues at distinct times in development. Precedence exists in the Ecdysone receptor, a Drosophila melanogaster hormone receptor involved in metamorphosis in which the isoforms are expressed in different combinations in different tissues during development (Talbot, et al ., Cell, 1993). Based on the genomic and cDNA structures of the
daf-12 gene, two regions are being tested for promoter activity to assess where the gene is expressed. One possible promoter region is 5 prime of the A1 isoform and the other is located in a 14 kbp intron that is upstream of the A2 and B isoforms. We have created transgenic animals carrying different fragments fused to the GFP and are currently determining their expression patterns.