Worm Breeder's Gazette 11(5): 77

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.

lin-25, a Gene Involved in Vulval Cell Fate Determination

Simon Tuck and Iva Greenwald

lin-25 gene activity is required for vulval development (Ferguson 
and Horvitz, 1985.  Genetics 110 17-72).  We are particularly 
interested in this gene because it may provide a link between two 
binary decisions during vulval precursor cell (VPC) fate 
specification: the vulva/non-vulval decision, controlled by let-60 
activity (Han and Sternberg, 1990.  Genetics 126.  899-913; Beitel et 
al., 1990.  Nature; 348, in press) and the 2 /non-2  decision, 
controlled by lin-12 activity (Greenwald et al., 1983.  Cell 34, 435-
444).  Two observations support this idea.  Firstly, in lin-25(e1446) 
hermaphrodites no VPCs adopt the 2  fate: P.6p undergoes the 1  fate 
and the other VPCs adopt the 3  fate (Ferguson et al., 1987.  Nature 
326.  259-267).  Secondly, double mutants carrying lin-25 loss of 
function mutations and either let-60 gain of function mutations or lin-
12 gain of function mutations suggest that lin-25 acts downstream of 
let-60 but upstream of lin-12 (P. Sternberg, personal communication 
and our own observations).
We have obtained evidence that lin-25(e1446) is a null allele.  We 
generated a deficiency in the region, arDf1, which fails to complement 
markers from vab-8 to lin-25 and used it in genetic dosage studies 
with lin-25(e1446), 45ts) and a new allele, lin-25(
ar90), which we isolated in a complementation screen.  The phenotype 
of lin-25(e1446) is similar both to that of lin-25(ar90) and to 
another lin-25 mutation, lin-25(n1063).  We plan to examine the null 
phenotype of lin-25 and the interactions of lin-25 null mutations with 
other mutations affecting vulval development in more detail.
We have performed temperature shift experiments on individual lin-25(
n545ts) hermaphrodites in which the developmental stage at the time of 
shifting was determined by Nomarski microscopy.  Our results extend 
those of Ferguson et al.  (1987) who performed temperature shift 
experiments on synchronized populations of lin-25(n545ts) 
hermaphrodites and concluded that the temperature-sensitive period (
TSP) for lin-25 synthesis or activity (required for egg-laying) 
extends from the mid-L2 stage to the mid-L3 stage.  lin-25(n545ts) 
animals are essentially 100% vulvaless at 25 C but are (>90%) egg-
laying competent at 15 C.  We found that some lin-25(n545ts) 
hermaphrodites shifted from 25 C to 15 C even as late as early L4 
stage (well after the VPCs had divided) were Egl+.  These results 
suggest that lin-25 activity is required not only for proper VPC fate 
specification, but also for another (as yet unknown) event that occurs 
during the L4 stage.  Interestingly, temperature shift experiments 
have revealed that lin-12 activity is also required in the early L4 
stage for egg-laying competence in addition to its earlier role in VPC 
fate specification (Meera Sundaram, personal communication).
In our efforts to clone lin-25 we have mapped Tc1 induced RFLPs in 
congenic strains kindly supplied by A.  Telfer.  These strains contain 
BO chromosomal DNA in the rol-4 to par-1 interval (in which lin-25 
maps) in an N2 background.  The RFLP closest to lin-25, which we have 
named arP1, maps 0.05mu to the right.  We have cloned the DNA flanking 
arP1 by inverse PCR and used it to isolate two lambda clones.  These 
clones were fingerprinted by Alan Coulson and John Sulston to the 
cosmid C06C1.  We have established a metric for the region of 44 
cosmid lengths/map unit by comparing the physical and genetic 
distances between arP1 and a second cloned polymorphism present in the 
congenic strains, TCPAR1.  Using the metric we estimate that lin-25 
lies on one of the cosmids immediately to the left of C06C1.  We are 
presently attempting to rescue the lin-25 mutant phenotype by 
microinjection experiments with these cosmids.