Molecular and functional studies of lag-2, a gene required for cell-cell interactions during development of the nematode C. elegans

In the nematode Caenorhabditis elegans, the glp-1 and lin-12 genes encode similar receptor proteins and are homologous to Notch of Drosophila. The GLP-1 and LIN-12 proteins appear to be functionally interchangeable. Therefore, GLP-1 and LIN-12 are likely to interact with similar or identical proteins. The strong loss-of-function phenotype of the lag-2 gene is virtually the same as that of lin-l2 glp-1 double mutants. The lag-2 gene has therefore been predicted to encode a protein common to the GLP-1 and LIN-12 signal transduction pathways.;We (Sam Henderson, I and Eric Lambie) cloned the lag-2 gene and I sequenced lag-2 genomic and cDNA clones. lag-2 encodes a membrane protein with structural similarity to Drosophila proteins Delta and Serrate (putative signaling ligands for Notch). I analyzed all available 16 loss-of-function lag-2 alleles and established the lag-2 null phenotype at the molecular level. I have also shown evidence that a family specific cysteine-rich DSL motif present in the predicted extracellular domain is crucial for LAG-2 function. In addition, I have shown that insertion into the N-terminal domain can cause dominant negative effects, implying that the N-terminal domain is also required for normal LAG-2 function. By using Green Fluorescent Protein reporter constructs, I have shown that lag-2 is expressed in the distal tip cell and its precursor cells throughout postembryonic gonadal development and LAG-2 may be associated with the cell membrane. I also demonstrated that expression of the signaling ligand LAG-2 in the signaling distal tip cell is independent of the receptor GLP-1 expression in the receiving tissue the germ line, representing yet another hallmark of the inductive interaction. Finally, I have demonstrated that the two homologous signaling ligands for the GLP-1/LIN-12 receptors, APX-1 and LAG-2, are functionally interchangeable, suggesting that the specificity of the GLP-1/LIN-12 signal transduction pathway does not rely on unique ligand/receptor interaction.