Worm Breeder's Gazette 14(1): 34 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Stanford University School of Medicine, Stanford University, Stanford, CA 94305.
Intercellular communication regulates the expression of the three cell fates (primary, secondary, and tertiary) during vulval development. The LIN-12 receptor is required for the specification of the secondary cell fate, presumably by lateral signalling between the primary cell (P6.p) and the presumptive secondary cells (P5.p and P7.p). We propose that P6.p expresses the lateral signal, yet the vulval lateral signal has yet to be identified. We have investigated whether LAG-2 could be the ligand for LIN-12 in vulval development. lag-2 was first isolated in a genetic screen due to its Lag (Lin and Glp) phenotype and encodes a protein similar to the putative ligands (APX-1, Delta and Serrate of Drosophila) of receptors related to LIN-12 (GLP-1 and Drosophila Notch) (Lambie and Kimble, 1991; Tax et al., 1994; Henderson et al., 1994). Furthermore, lag-2 is expressed at the right time and in the right cells to be the ligand for both GLP-1 and LIN-12 receptors in non-vulval inductive events (Henderson et al., 1994; Wilkinson et al., 1994). Homozygous lag-2 null mutants die as early L1 larvae and have defects in specification of certain cell fates. This early lethality prevented the observation of any possible lag-2 vulval defects. Therefore, we used mosaic analysis to test whether lag-2 was required for proper vulva formation. We can observe any lag-2 vulval phenotypes by identifying animals that lack lag-2(+) activity in the Pn.p cells, but maintain lag-2(+) activity in cells essential for viability. We constructed the lag-2 mosaic strain by injecting unc-29(e1072) I; ncl-1(e1942) III; unc-30(e190) IV; lag-2(q411)/+ V animals with the following DNAs that contain the following four genes: unc-29(+) (cosmid C45D10), ncl-1(+) (cosmid C33C3), unc-30(+) (plasmid pUnc30), and lag-2(+) (plasmid pJK254). We isolated a transgenic line that was homozygous for lag-2(q411) and transmitted the array to approximately 50% of the progeny. Using the Unc-29, Unc-30 and Ncl (an enlarged nucleolus) phenotypes to screen for mosaic animals, we identified eight animals that lacked lag-2 in all six Pn.p cells due to a loss of the array in either AB or ABp. All eight of these animals developed a normal vulva as determined by the number, position and morphology of vulval nuclei during the fourth larval stage. This result indicates that P5.p and P7.p express the secondary cell fate when lag-2(+) activity is absent in the Pn.p cells, and suggests that LAG-2 does not act as the lateral signal. Alternatively, LAG-2 may act as a LIN-12 ligand in the Pn.p cells but concomitant expression of other lag-2/apx-1 homologs during vulval induction can substitute for lag-2 function. Although lag-2 is not required for lateral signalling, it may have a role in vulval morphogenesis since approximately 20% of the animals in the mosaic strain exhibit an Egl phenotype. The Egl phenotype does not appear to be caused by defective vulval cell lineages since two of the eight mosaics described above seemed to have normal vulval cell lineages but were Egl. This Egl phenotype also does not appear to be caused by defects in sex muscle migration or attachment to the vulval cells, as phalloidin staining of the sex muscles did not reveal any abnormalities. This Egl phenotype could be similar to the "late defect" in vulval morphogenesis seen in loss-of-function lin-12 mutants (Sundaram and Greenwald, 1993). This strain could be used to map lag-2 lethal foci (cells that require lag-2 and are essential for viability). A lethal focus likely does not exist in the AB lineage since we found three animals that lost the array in AB and were viable. However, at least one lethal focus likely exists in cells derived from the P1 lineage because we never observed a loss in the P1 lineage in over 200 potential mosaic animals screened. In addition to lethality, this mosaic strain is a potential tool that could be used to investigate the effects of loss of lag-2(+) activity in specific cells. We would like to thank the Kimble lab for their generous donations of the lag-2 strains and DNAs. Henderson, S. T., D. Gao, E. J. Lambie, and J. Kimble (1994b) lag-2 may encode a signaling ligand for the GLP-1 and LIN-12 receptors of C. elegans. Development 120, 2913-24 Lambie, E. J. and J. Kimble (1991) Two homologous regulatory genes, lin-12 and glp-1, have overlapping functions. Development 112, 231-40 Sundaram, M. and I. Greenwald (1993) Genetic and phenotypic studies of hypomorphic lin-12 mutants in Caenorhabditis elegans. Genetics 135, 755-63 Tax, F. E., J. J. Yeargers, and J. H. Thomas (1994) Sequence of C. elegans lag-2 reveals a cell-signalling domain shared with Delta and Serrate of Drosophila. Nature 368, 150-4 Wilkinson, H. A., K. Fitzgerald, and I. Greenwald (1994) Reciprocal changes in expression of the receptor lin-12 and its ligand lag-2 prior to commitment in a C. elegans cell fate decision. Cell 79, 1187-98