Figure 4. DAF-12 regulates LIT-1 gene expression in C. el- egans. In A-C, the wild-type or
m11m31 mutant 4.2 fragment was subcloned upstream of a minimal
pes-10 promoter driving GFP expression and microinjected into wild-type or
daf-12 mu- tant
rh61rh412 C. elegans. (A) GFP expression in the pharynx (arrows) in N2 worms with wild-type 4.2. (B) Absence of GFP pharyngeal expression in N2 worms injected with the 4.2m11m31 mutant reporter. One-hundred percent of animals exhibited the effect. (C) Reduced GFP expression in
daf-12 mu- tant worms (arrows) injected with wild-type 4.2 fragment. In four independent injections, >90% of animals exhibited the ef- fect in three cases; in the fourth, 50% displayed the reduction. (D-L) A 4-kb LIT-1 genomic segment (nucleotides 4527-8561 from cosmid W06F12, with upstream/intronic sequences in red and exons in blue) with the LIT-1b initiating methionine codon (M) at its midpoint, and bearing either the wild-type or
m11m31 4.2 segment in its second intron (nucleotides 7201-7408), was fused in frame to GFP and microinjected into wild-type or daf- 12 mutant
rh61rh412 C. elegans. (D-F) LIT-1-GFP expression in pharynx (arrows) with wild-type or
m11m31 4.2 constructs in L4 N2 worms, or with wild-type 4.2 constructs in
daf-12 mu- tant worms, respectively. In E, 100% of animals had no pharyn- geal GFP expression; in F, >90% of animals had reduced GFP expression in pharynx. (G) Low levels of LIT-1-GFP expression from 4.2 construct in seam cells of L4 N2 animals (arrow); GFP expression in embryos served as positive control. (H,I) Elevated LIT-1-GFP expression in seam cells (arrows) with
m11m31 4.2 constructs in L4 N2 worms, or with wild-type 4.2 constructs in
daf-12 mutant worms, respectively. Five times more animals than in G had elevated expression. (J) Low levels of LIT-1-GFP expression from 4.2 construct in vulva cells (arrow) of L4 N2 animal. (K,L) Elevated LIT-1-GFP expression in vulva cells (ar- rows) with
m11m31 4.2 constructs in L4 N2 worms, or with wild-type 4.2 constructs in
daf-12 mutant worms, respectively. Five times more animals than in J had elevated expression. In A-L, >50 animals per panel were examined. In A-F and J-L, images were taken at 1000×; in G-I, images were taken at 400×. (M) Regulation of endogenous LIT-1 expression by DAF-12 in L4. LIT-1a and LIT-1b mRNAs were analyzed by qRT PCR; data were normalized to AMA-1 abundance values, which were as- signed a value of 100. CAP-1, CAP-2, and RGR-1 were used as controls; various other control gene transcripts, including ACT- 5, RPL-19, and DPY-26, also did not change (data not shown).