Laser ablation of a single cell, ABplppppp, can abolish rectal formation in C. elegansembryos. This cell is one of three ABp sublineages (with ABplpappp and ABprppppp) that express
pal-1at the 250-cell stage(Edgar et al,Dev Biol 229:71,2001). Rescue of the
pal-1null mutation
ct224, which has a Nob phenotype, with a genomic construct containing only 1.1 kb of 5' sequence gives nonviable larvae without a rectum, a phenotype resembling mutants with defects in the Lag (
lin-12 glp-1) signalling pathway (Lambie and Kimble, Dev 112:231,1991). In
lag-1 and
lag-2 mutants, a
pal-1::GFP reporter with 7 kb of 5' sequence is not expressed in the AB lineage, although normally in other lineages. That difference suggests that
pal-1 expression in this specific lineage is initiated by a Lag signal. There are 6 binding sites for Lag-1, the final transcription factor in the Lag pathway, within 7 kb upstream of the
pal-1 coding start (designated -5.5,-3.5,-2.3,-2.1,-1.3,-0.1), and 4 intragenic sites, representing consensus sequences of both C. elegans (Christensen et al, Dev 122:1379,1996) and the Drosophila homolog SuH (Bailey and Posakony, Genes and Dev 9:2609,1995). Deletions of the internal or the -5.5 putative binding sites did not affect reporter expression in the AB lineage, while more proximal 5' deletions eliminated it. Site-directed mutagenesis of the five 5' binding sites in a construct with 5 kb 5', singly or in combination, weakened or eliminated AB reporter expression. The most important sites are -3.5, -2.3, and -1.3. Sites appear to act cooperatively. Reporters with multiple mutations also consistently showed ectopic expression in 4 additional head cells of the 300-cell embryo (lineage identification pending). We are currently addressing the question of which of the possible ligands (Lag-2 or Apx-1) and receptors (Lin-12 and/or Glp-1) are the operative signaling molecules, and defining the possible signaling cells. Lag-2 appears the most likely ligand, as it is expressed in MSapa and app, in the left-side MS lineage, which contact ABplpappp and plppppp at the 100-cell stage, one cell division before the ABs express
pal-1. Because the two left-side AB cells express
pal-1 well before ventral cleft closure, and the right homolog ABprppppp expresses only after the cells meet at the ventral midline and have divided, we think that ABprpppppa/p may require an inductive signal from their left-side homologs to initiate
pal-1 expression. We plan to characterize reporter expression on the right side when such contact is delayed or prevented.