Worm Breeder's Gazette 13(2): 49 (February 1, 1994)

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.

The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell

Sam Henderson, Dali Gao, Eric Lambie and Judith Kimble

The germline of C. elegans provides a simple system to investigate cell-cell
signaling in a multicellular organism. The germline consists of reflexed tube, with
mitotically proliferating cells at one end (distal end) and differentiated gametes at the
other (proximal). At the very distal end of the germline is a single cell, known as the
distal tip cell (DTC). Continual proliferation of germ cells requires the presence of the
DTC, and the product of the glp-1 gene. It has been shown that glp-1 is not required in
the DTC and has the molecular structure consistent with it being a receptor protein. It
is likely that the GLP- 1 protein is functioning in the germline to receive a signal from
the DTC (for review see Lambie and Kimble, 1991a). The lag genes ( lag-1 and lag-2
)have phenotypes identical to lin-12 / glp-1 double mutants. These genes are thought
to function in both the lin-12 and glp-1 signaling pathways (Lambie and Kimble, l991b
).Weak alleles of the lag-2 gene show a phenotype similar to that of loss of function
glp-1 alleles. This suggests that lag-2 is functioning in the pathway that regulates
germ line proliferation. To better understand the role of lag-2 in controlling germline
proliferation we have cloned the lag-2 gene and examined its expression pattern.
  We cloned lag-2 using PCR deficiency mapping and microinjection rescue.
Several cDNA clones were isolated and sequenced from embryonic cDNA libraries
constructed by P. Okkema (Okkema et al. WBG#12 pl6 )and J. Ahringer. The
sequence has similarity to the Drosophila gene Delta and is probably identical to the
lag-2 gene reported by F. Tax and Jim Thomas (1993 meeting abstract). The predicted
protein is 416 amino acids long, and contains a signal sequence, three cysteine rich
regions, a single transmembrane domain, and an intracellular PEST sequence. The
first cysteine rich region is similar to a region found in both the Drosophila genes Delta
and Serrate. The second cysteine rich region contains 6 cysteines that do not conform
to any known motif, but look most similar to two half EGF repeats. The third cysteine
rich region matches the consensus EGF repeat motif. The predicted intracellular
domain is 109 amino acids long and contains a good PEST sequence. Four loss of
function lag-2 alleles were examined by SSCP PCR and were found to have changes
within the coding region of the presumptive lag-2 gene, including two nonsense
mutations.
  To look at the expression of the lag-2 gene, two lacZ fusion constructs were made
using pPD21 .28(Fire, et al. 1990). The first construct, lag-2 5':: lacZ , fuses the 5'
flanking region of lag-2 to a 337-galactosidase gene equipped with a nuclear localization
signal. The second construct, lag-2 ::lacZreplaces the intracellular portion of lag-2
with 337-galactosidase. Adult worms carrying lag-2 5':: lacZ show X-gal staining only
in the nucleus of the DTC, and those carrying lag-2 ::lacZshow staining primarily in
the DTC. Since extrachromosomal arrays are poorly expressed in the germline, we
used in situ hybridization (Evans et al. this WBG) to examine lag-2 expression in
adult germlines. In situ hybridization showed that lag-2 RNA is exclusively located in
the DTC and not in the germline. Both in situ and X-gal staining results are
consistent with the model that LAG-2 functions as a ligand for the putative GLP-l
receptor in the germline.
 Lambie, E. and Kimble, J. 1991a. Genetic control of cell interactions in nematode development.
Annu. Rev. Genet. 1991 25:411-436.
 Lambie, E. and Jimble, J. 1991b. Two homologous regulatory genes, lin-12 and glp-1 ,have
overlapping functions. Development 112:231-240.
 Fire, A. Harrison, S.W., and Dixon, D. 1990. A modular set of lacZ fusion vectors for studying
gene expression in Caenorhabditis elegans. Gene 93:189-198.