Chemosensation of dauer pheromone is mediated by two parallel genetic pathways, corresponding to group 1 and group 2 Daf-c genes. We are investigating the role of group 2 Daf-c genes (
daf-1, -4, -8, -7 and -14) in this process at molecular and cellular level. Molecular identities of four of these genes have been reported by Don Riddle's lab.
daf-7 encodes a homolog of TGF-b(1),
daf-1 and
daf-4 encode homologs of TGF-b receptors(2,3), and
daf-8 encodes a homolog of the Drosophila gene Mad (Mothers against dpp)(4). We have cloned
daf-14 and found that it is also has homology to Mad. These genes are members of a family(5) that includes Mad from Drosophila(6) and
sma-2,
sma-3,
sma-4(5),
daf-8 and
daf-3(7) from C. elegans. All of these genes are implicated in TGF-b related signal transduction, suggesting they play a conserved role. Known members of this gene family contain two conserved regions, DH1 and DH2(5). Genefinder analysis and sequence alignment of the
daf-14 genomic DNA predicts a protein with strong similarity to the DH2 region but without a DH1-like domain. Also, no homology to DH1 was detected in a search of genomic sequence upstream of the
daf-14 DH2 region. All three mutations in
daf-14 disrupt highly conserved residues in the C terminal half of DH2 region. This suggests that
daf-14 is unique among Mad related genes in not having a DH1 region. However, because the mRNA structure has not been confirmed, we cannot yet rule out the possibility that the DH1 domain is present. We generated two integrants of
daf-14(+)-carrying transgenic arrays, which probably overexpress the
daf-14 gene product. These arrays suppress the Daf-c defect of
daf-14 and
daf-8, but not
daf-1, -4 and -7. These results are consistent with many models, including epistatic interactions and functional substitution of
daf-14 for
daf-8. To begin to determine the cellular basis for TGF-b signaling in the dauer pathway, we generated a GFP fusion to
daf-7. The transgene is expressed in the two ASI amphid neurons during larval development. The expression is reduced at higher temperatures and in the presence of dauer pheromone. These findings are consistent with cell-kill results that indicated that ASI cells function to repress dauer formation(8). We suggest that ASI synthesizes
daf-7 in the absence of pheromone to repress dauer formation. Expression study of
daf-14 using a GFP fusion is currently underway. 1. Lim et al., WM93 abstract
p15, Lim, Ph.D. Thesis, University of Missouri, Columbia, Missouri. 2. Georgi et al., Cell 61 635-645 3. Estevez et al., Nature 365 644-649 4. Estevez and Riddle, WBG14.2
p37 5. Savage et al., PNAS USA 93 790-794 6. Sekelsky et al., Genetics 139 1347-1358 7. Koweek et al., ECWM96 abstract. 8. Bargmann and Horvitz, Science 251 1243-1246, W.S.S. and J.H.T. unpublished results.