lin-48∷gfp transgenes from C. elegans and C. briggsae tested in each species indicate only the C. elegans gene in C. elegans animals is expressed in the excretory duct cell. All other expression patterns are conserved. The 2.4 kb of Ce-lin-48 upstream regulatory sequences is indicated with a solid line and white blocks. The 3.0 kb of Cb-lin-48 upstream regulatory sequences is indicated by a broken line and black blocks. Blocks correspond to conserved DNA sequences in the area of the previously defined regulatory element lre2 and two additional regions more proximal to the transcriptional start site.

Potential CES-2-binding sites in Ce-lin-48 promoter. (1) DNA sequence of ∼60 bp Ce-lin-48 proximal region. CES-2-binding sites are shown by arrows under the sequences. (2) Alignment between the CES-2-binding sites in the Ce-lin-48 proximal region, ces-1 upstream element (Metzstein and Horvitz 1999), and the CES-2/PAR family consensus (Metzstein et al. 1996). Y = T or C; R = A or G.

lin-48 expression and function in C. elegans and C. briggsae. (a) Nomarski DIC micrograph of wild-type L3 C. elegans animal showing the excretory system. (b) Epi-fluorescent micrograph of animal in a, showing expression of Ce-lin-48∷gfp. The location of the excretory cell and excretory duct cell are indicated by an asterisk and an arrow. The excretory duct is indicated by an arrowhead. The excretory duct forms more posterior in wild-type C. elegans (c) than in C. elegans lin-48 mutants (d) or C. briggsae (e). ces-2 mutant animals exhibit the same excretory duct cell morphological phenotype as lin-48(sa469) mutants. Bracket shows distance between the duct and the base of the posterior pharynx. (f) lin-48 mutant males develop abnormal ectopic spicule cells anterior to the normal spicule cells. (g) lin-48 mutant animal rescued by Cb-lin-48 genomic clone, resulting in development of normal spicule cells. Arrows indicate cells expressing spicule cuticle. Scale bars, 10μm. (Left) anterior; (top) dorsal.

(1) Experiments to identify cis-regulatory sequences important for excretory duct cell expression in C. elegans. (a) A mutation in the Ce-lin-48 promoter (indicated by X) shows the conserved lre2 sequence is important for expression in both excretory duct and hindgut cells (data from Johnson et al. 2001). (b–j) Chimeric promoter sequences resulting from DNA swap between C. elegans and C. briggsae and deletion analysis of the chimeric promoter identify at least four additional regulatory regions that contribute to Ce-lin-48 expression in the duct cell. (k) Mutations in putative CES-2-binding sites in the Ce-lin-48 promoter reduced the excretory duct cell expression. (2) Experiments to show that ces-2 affects lin-48 excretory duct cell expression. (l–s) ces-2(RNAi) reduced lin-48 expression in transgenic animals. Reduction of lin-48 expression by transgenes marked with asterisk was also confirmed in ces-2(n732) mutant background. The percentages of cells expressing GFP (black bar), expressing very low, but detectable levels of GFP (gray bar) or not expressing GFP (white bar), are indicated for each construct. (n) Number of animals scored for expression. Source of DNA in transgene indicated as in Figure 2. For chimeric clones, arrowhead indicates the point of the DNA swap. GFP expression in hindgut was used as an internal control for expression level and presence of the transgene in all experiments.